ijBRAKl 
gTATE  PLAN?  BOAR» 


Technical  Series,  No.  14. 

U.  S.  DEPARTMENT   OF   AGRICULTURE, 

BUREAU    OF    ENTOMOLOGY. 
L.  0..  HOWARD,  Entomologist  and  Chief  of  Bureau. 


THE 


BACTERIA  OF  THE  APIARY: 

WITH  SPECIAL  REFERENCE  TO 
BEE  DISEASES. 


BY 

GERSHOM  FRANKLIN  WHITE,  Ph.  D., 

Expert  in  Animal  Bacteriology,  Biochemic  Division,  Bureau  of  Animal  Industry. 


Issued  November  6,  1906. 


S3 

mi 


WASHINGTON: 

GOVERNMENT    PRINTING    OFFICE. 
1900. 


BUREAU  OF  ENTOMOLOGY. 

L.  O.  Howard.  Entomologist  and  Chief  of  Bureau. 

C.  L.  Marlatt.  Entomologist  and  Acting  Chief  in  absence  of  Chief. 

\l.  S.  Clifton,  Chief  clerk. 

F.  H.  Chittenden,  in  Charge  of  breeding  experiments. 

A.  D.  Hopkins,  in  charge  of  forest  insect  Investigations. 

W.  D.  Hunter,  in  charge  of  cotton  boll  weevil  investigations. 

F.  M.  Webbteb,  in  charge  of  cereal  and  forage-plant  insect  investigations. 
A.  L.  Quaintance.  in  charge  of  deciduous-fruit  insect  investigations. 

D.  M.  Rogers,  in  charge  of  gipsy  and  brown-tail  moth  work. 
A.  W.  Morrill,  engaged  in  white  fig  in  rest igat ions. 

E.  S.  G.  Titus,  in  charge  of  gipsy  moth  laboratory. 
C.  J.  Gilliss,  engaged  in  silk  investigations. 

R.  P.  Currie,  assistant  in  charge  bf  editorial  work. 
Mabel  Colcord,  librarian. 


Apicultueal  Investigations. 

Frank  Benton,  in  charge  (absent). 

E.  F.  Phillips,  acting  in  charge. 

J.  M.  Rankin,  in  charge  of  apicultural  station,  Chico,  Cal. 

Jessie  E.  Marks,  apicultural  clerk. 


Digitized  by  the  Internet  Archive 
in  2013 


http://archive.org/details/bacteriaofapiaryOOwhit 


Technical  Series,  No.  14. 

U.  S.  DEPARTMENT  OF   AGRICULTURE, 

/ 

BUREAU    OF1    ENTOMOLOGY. 
L.   0.   HOWARD,  Entomologist  and  Chief  of  Bureau. 


THE 

BACTERIA  OF  THE  APIARY, 

WITH  SPECIAL  REFERENCE  TO 
BEE  DISEASES. 

BY 

GERSHOM  FRANKLIN  WHITE,  Ph.  D., 

Expert  in  Animal  Bacteriology,  Biochemic  Division,  Bureau  of  Annual  Industry. 


Issued  November  6,  1906. 

t 


WASHINGTON: 

GOVERNMENT    PRINTING    OFFICE. 
1906. 


LETTER  OF  TRANSMITTAL. 


U.  S.  Department  of  Agriculture, 

Bureau  of  Entomology, 
Washington,  D.  6'.,  Septt  mber  &£,  1906. 

Sir:  I  have  the  honor  to  transmit  the  manuscript  of  a  paper  on 
the  bacteria  of  the  apiary,  with  special  reference  to  bee  diseases,  by 
Dr.  G.  F.  White,  expert  in  animal  bacteriology  in  the  Biochemic 
Division  of  the  Bureau  of  Animal  Industry.  This  paper  was  pre- 
pared by  Doctor  White,  as  a  thesis  in  part  fulfilment  of  the  require- 
ments for  the  degree  of  doctor  of  philosophy,  at  Cornel]  University, 
in  June,  1905.  The  Bureau  of  Entomology  considers  itself  fortu- 
nate in  obtaining  it  for  publication,  since  in  this  way  a  wider  distri- 
bution can  be  made  than  would  be  possible  were  it  published  in  a 
journal  devoted  exclusively  to  bacteriological  investigations.  It  is 
hoped  that  the  publication  of  these  fact-  may  help  to  clear  up  the 
confusion  which  now  e\i>ts  concerning  the  causes  of  the  two  most 
common  diseases  of  the  brood  of  bees.  I  recommend  that  the  manu- 
script be  published  as  Technical  Series,  No.  14,  of  this  Bureau. 

Doctor  White  wishes  to  acknowledge  his  indebtedness  to  Dr. 
Veranus  A.  Moore,  professor  of  comparative  pathology  and  bac- 
teriology of  Cornell  University,  under  whose  direction  this  work 
was  done;  to  Dr.  E.  F.  Phillips,  acting  in  charge  of  apiculture, 
Bureau  of  Entomology,  United  States  Department  of  Agriculture, 
for  encouragement  and  assistance  in  the  preparation  of  this  manu- 
script; and  to  Messrs.  Mortimer  Stevens,  Charles  Stewart,  N.  D. 
West,  and  W.  D.  Wright,  bee  inspectors  of  the  State  of  New  York, 
for  their  interest  in  the  work. 

Eespectfully,  L.  O.  Howard, 

Entomologist  and  Chief  of  Bureau. 

Hon.  James  Wilson, 

Secretary  of  Agriculture. 

2 


PREFACE. 


The  spread  of  diseases  of  the  brood  of  bees  is  to-day  a  great  menace 
to  the  bee-keeping  industry  of  the  United  States.  It  is  therefore  of 
great  importance  that  all  phases  of  these  diseases  should  be  investi- 
gated as  thoroly  as  possible,  and  this  paper,  it  is  believed,  will  help 
in  clearing  up  some  disputed  points  in  regard  to  the  cause  of  the  two 
most  serious  brood  diseases. 

Dr.  G.  F.  White  has  offered  this  paper  for  publication  as  a  bulletin 
in  the  Bureau  of  Entomology  because  in  that  way  the  statements 
herein  contained  may  become  more  widely  known  than  would  be  the 
case  were  it  published  in  some  journal  devoted  exclusively  to  bacteri- 
ological investigations.  Obviously  there  are  many  points  still  un- 
settled, and  it  is  hoped  that  some  of  these  may  be  taken  up  for  in- 
vestigation in  the  near  future,  but  the  results  so  far  obtained  should 
by  all  means  be  made  known  to  the  persons  practically  engaged  in 
bee  keeping. 

The  necessity  for  the  study  of  nonpathogenic  bacteria  found  in 
the  apiary  may  not  be  at  first  evident  to  the  ordinary  reader.  When 
it  is  seen,  however,  that  some  of  the  investigators  of  bee  diseases  have 
apparently  mistaken  Bacillus  A  or  some  closely  allied  species  for 
Bacillus  alvei  it  will  be  evident  that  a  study  of  nonpathogenic  germs 
is  necessary  to  a  thoro  investigation  of  the  cause  of  these  diseases  and 
a  full  understanding  of  the  confusion  which  has  existed. 

The  names  which  should  be  used  for  the  diseased  conditions  of 
brood  was  a  matter  which  arose  after  this  paper  was  offered  for  pub- 
lication. It  was  desired  that  out  of  the  chaos  of  names  in  use  cer- 
tain ones  be  chosen  which  would  be  distinctive  and  still  clear  to  the 
bee  keepers  who  are  interested  in  work  of  this  nature.  Unfortu- 
nately, after  a  short  investigation.  Dr.  W.  R.  Howard,  of  Fort 
Worth,  Tex.,  gave  the  name  "  Xew  York  bee  disease,"  or  "  black 
brood,"  to  a  disease  which  Cheshire  and  Cheyne  described  in  1885  as 
"  foul  brood."  Since  this  is  the  disease  in  which  Bacillus  alvei  is 
present,  we  can  not  drop  the  name  "  foul  brood,"  and  the  word 
"  European  "  is  used  to  distinguish  it  from  the  other  disease.  The  bee 
keepers  of  the  United  States  have  been  taught  that  the  type  of  brood 
disease  characterized  by  ropiness  of  the  dead  brood  is  true  foul  brood, 

3 


4  PREFACE. 

but  since  Bacillus  alvei  is  not  found  in  this  disease  it  obviously  is  not 
the  same  disease  as  that  described  by  Cheyne.  It  would  be  well-nigh 
impossible,  however,  to  change  the  name  of  this  disease,  and  any  effort 
in  that  direction  would  merely  result  in  complicating  laws  now  in  force 
which  control  the  infectious  diseases  of  bees  and  would  serve  no  good 
purpose.  This  disease  is  here  designated  "American  foul  brood." 
These  names  have  been  chosen  only  after  consultation  with  some  of 
the  leading  bee  keepers  of  the  United  States,  and  these  distinguishing 
terms  were  chosen  by  the  majority  of  those  consulted  as  indicating 
the  place  in  which  the  diseases  were  first  investigated  in  a  thoroly 
scientific  manner.  Both  diseases  are  found  in  Europe,  as  well  as  in 
America,  so  that  the  names  indicate  nothing  concerning  the  geo- 
graphical distribution  of  the  maladies. 

Strangely  enough,  certain  writers  for  our  American  apicultural 
papers  have  seen  fit  to  take  exception  to  some  of  the  statements  made 
in  this  paper  without  having  first  found  out  the  reasons  for  the  de- 
cisions herein  published.  Apiculture  will  not  be  advanced  to  any 
appreciable  extent  by. such  eagerness  to  rush  into  print,  especially 
when  there  is  not  a  semblance  of  scientific  investigation  back  of  the 
criticism. 

E.  F.  Phillips, 
Acting  hi  Charge  of  Apiculture. 


CONTENTS. 


Page. 

Introduction    7 

Technique    7 

Obtaining  material  for  study . 7 

Obtaining  cultures 7 

Differentiation  and  identification  of  bacteria 9 

The  cultures  which  are  described 9 

Morphology,  staining  properties,  and  oxygen  requirements,  with  sug- 
gestions on  variations 9 

Media  employed  and  suggestions  as  to  the  description  of  cultures 10 

PART    I.       BACTERIA    OF    THE    NORMAL    APIARY. 

Bacteria  from  the  combs 13 

Bacteria  from  pollen 15 

Bacteria  in  honey  and  normal  larvae 1G 

Bacteria  upon  the  adult  bees 16 

Bacteria  of  the  intestine  of  the  healthy  honey  bee 18 

Saccharomyces  and  fungi 25 

Tabulation  of  micro-organisms  normally  present  in  the  apiary 28 

Summary  to  Part  I 29 

Bibliography  to  Part  I "   29 

PART    II.    THE    DISEASES    OF    BEES. 

Brief  history 30 

The  term  "  foul  brood"  as  hitherto  applied 31 

European  foul  brood  (foul  brood  of  Cheyne) 32 

Symptoms    32 

Confusion  regarding  foul  brood  in  America 33 

The  present  investigation 34 

Bacillus-    alvei 36 

Inoculation    experiments 37 

Distribution  of  Bacillus  alvei  in  infected  hives 38 

Experiments  with  formaldehyde  gas 39 

American  foul  brood 40 

Symptoms    " • 40 

The  present  investigation 41 

Bacillus    larvae 42 

The  so-called  "pickle  brood" 43 

The  so-called  "black  brood" 43 

Palsy  or  paralysis 44 

Summary  to  Part  II : 44 

Conclusions     45 

Bibliography  to  Part  II 46 

Index 47 

5 


THE  BACTERIA  OF  THE  APIARY  WITH  SPECIAL 
REFERENCE  TO  BEE  DISEASES. 


INTRODUCTION. 

Since  bacteriology  is  one  of  the  youngest  of  the  sciences,  it  is  only 
natural  that  there  should  be  many  problems  concerning  which  there 
is  much  confusion,  and  many  others  concerning  which  nothing  is 
known.  In  a  study  of  the  saprophytic  bacteria  this  is  especially 
true;  the  exploration  of  this  jungle  of  micro-organisms  is  scarcely 
begun.  Comparatively  few  species  have  been  studied  and  named, 
and  a  much  less  number  can  be  identified.  From  studies  that  have 
been  made  one  is  led  to  believe  that  the  species  which  might  be 
classed  under  bacteria  outnumber  by  far  all  the  macroscopic  plants 
known.  Comparatively  little  is  as  yet  known  concerning  the  dis- 
tribution of  these  minute  organisms  in  nature,  their  needs  for  multi- 
plication and  growth,  their  power  of  endurance,  their  relations  the 
one  to  the  other,  their  relations  to  man  and  industries,  and  their 
relation  to  pathogenic  species.  Both  from  the  standpoint  of  scien- 
tific interest  and  from  the  standpoint  of  practical  economy  these 
problems  call  for  further  investigation. 

By  far  the  greatest  amount  of  work  which  has  been  done  in  the 
science  of  bacteriology  has  been  prompted  by  the  direct  or  indirect 
economic  importance  of  the  question.  This  is  largely  true  of  the 
present  investigation,  since  honey  bees  suffer  from  a  number  of 
diseases,  some  of  which  are  considered  in  Part  II. 

TECHNIQUE. 

Obtaining  Material  for  Study. 

If  necessary,  bees  may  be  conveniently  shipped  alive  by  mail  in 

cages  constructed  for  that  purpose.     Combs  also  may  be  sent  by  mail 

in  small  boxes.    If  combs,  honey,  pollen,  or  larvae  are  desired,  the  hive 

must  be  entered.    In  case  older  adult  bees  are  wanted  it  is  not  difficult 

to  supply  the  needs  from  the  entrance  to  the  hive.    To  capture  them 

one  may  stand  at  the  entrance  and  catch  the  unwary  toiler  as  she 

7 
9583— No.   14—06  M 2 


8  THE    BACTEBIA    OF    THE    APIARY. 

comes  in  loaded  with  pollen  and  honey.  After  the  victim  alights  on 
the  entrance  board,  by  the  aid  of  a  pair  of  forceps,  before  she  disap- 
pears within,  one  can  easily  lodge  her  safely  in  a  petri  dish.  It  is, 
however,  an  advantage  to  study  the  young  adult  bees  as  well  as  the 
older  ones,  and  if  young  ones  are  desired  they  may  be  taken  from 
the  combs  or  from  the  front  of  the  hive,  near  the  entrance. 

Obtaining  Cultures. 

(a)  From  combs, — With  sterile  forceps  small  pieces  of  the  comb 
are  put  directly  into  gelatin  or  agar  for  plates  or  incubated  in  bouil- 
lon for  24  hours  and  then  plated.  Growing  in  bouillon  and  plat- 
ing on  gelatin  is  usually  preferable. 

(b)  From  poUen. — The  same  technique  is  used  as  for  combs,  but 
the  direct  inoculation  of  gelatin  tubes  for  plates  i-  generally  pre- 
ferable. 

(c)  From  honey. — With  sterile  loops  honey  is  taken  from  uncapped 
and  capped  cells.  The  caps  are  removed  with  sterile  forceps  and  the 
honey  is  plated  directly  on  gelatin  or  agar.  Bouillon  tubes  are  in- 
oculated also  with  varying  quantities  of  the  honey. 

(d)  From  hi, -rti . — The  larva  is  carefully  removed  to  a  sterile  dish, 
and  with  sterile  scissors  the  body  i>  opened  and  the  contents  plated 
directly,  or  bouillon  cultures  are  firsl  made  and  later  plated,  if  a 
growth  a | >] tear-. 

(<  )  From  parts  of  th<  adult  bee,— In  studying  the  adult  bee,  a 
small  piece  of  blotting  paper  wet  with  chloroform  is  slipt  under 
the  cover  of  the  petri  dish  in  which  the  insects  have  been  placed,  and 
in  a  short  time  the  bees  are  under  the  influence  of  the  anesthetic. 
Then  with  sterile  scissors  a  leg.  a  wing,  the  head,  the  thorax,  or  the 
abdomen,  the  intestine  being  removed,  is  placed  in  bouillon  and,  after 
24  hours  incubation,  plated,  preferably  on  gelatin. 

When  it  is  desired  to  make  a  study  of  the  bacteria  of  the  intestine, 
the  intestinal  tract  is  removed  and  studied  as  follows:  The  bee  is 
flamed  and  held  in  sterile  forceps.  With  another  sterile  pair  of  for- 
ceps the  tip  of  the  abdomen  is  seized  and.  by  pulling  gently,  the  tip 
and  the  entire  intestine  are  easily  removed.  This  can  then  be  plated 
directly.  If  gelatin,  which  i>>  preferable,  is  used,  the  intestine  itself 
must  not  be  left  in  the  gelatin  or  the  medium  will  become  liquefied 
by  the  presence  of  the  tissue.  If  one  desires  to  obtain  cultures  of  the 
anaerobe,  which  is  quite  common  in  the  intestine,  it  is  most  easily 
obtained  in  pure  culture  by  the  use  of  the  deep  glucose  agar  (Liborius's 
method).  Cover  glass  preparations  made  direct  from  the  Avails  of 
the  intestine  or  its  contents  give  one  some  idea  of  the  great  number  of 
bacteria  frequently  present. 


MORPHOLOGY,  STAINING    PROPERTIES,  ETC.  \) 

Differentiation  and  Identification  of  Bacteria. 

These  very  low  forms  of  plant  life  show  a  marked  susceptibility  to 
environmental  conditions  and  those  desirous  of  speculating  on  prob- 
lems in  evolution  may  find  here  food  for  thought  and  experimenta- 
tion. On  account  of  this  susceptibility,  various  cultures  which  belong 
to  the  same  species  may  possess  slight  variations  in  some  one  or  more 
specific  characters.  Consequently  one  can  not  say  that  a  species  must 
possess  certain  definite  characters  and  no  others.  It  is  convenient, 
then,  to  think  of  a  species  as  more  or  less  of  a  group  of  individuals 
whose  characters  approximate  each  other  very  closely. 

In  this  paper  are  described  a  number  of  species  each  of  which,  in 
fact,  represents  a  group,  the  individual  cultures  of  which  approxi- 
mate each  other  so  closely  in  character  that  the  differences  may  be 
easily  attributed  to  environmental  conditions  which  are  more  or  less 
recent. 

Concerning  the  identification  of  species,  the  conditions  have  been 
well  summed  up  by  Chester.     He  says: 

Probably  nine-tenths  of  tbe  forms  of  bacteria  already  described  might  as  well 
be  forgotten  or  be  given  a  respectful  burial.  This  will  tben  leave  comparatively 
few  well-defined  species  to  form  tbe  nuclei  of  groups  in  one  or  another  of  wbicb 
we  shall  be  able  to  place  all  new  sufficiently  described  forms. 

The  variations  which  occur  and  the  very  incomplete  descriptions 
which  can  be  found  make  it  impossible  to  identify  many  species  even 
to  a  more  or  less  restricted  group.  For  these  reasons  some  of  the 
cultures  are  not  identified  or  named,  but  letters  are  used  for  conven- 
ience in  this  paper  to  represent  the  specific  part.  Migula's  classifica- 
tion has  been  used. 

The  Cultures  Which  are  Described. 

Plate  cultures  were  observed  for  some  weeks,  the  different  kinds  of 
colonies  which  appeared  being  especially  noted.  Subcultures  were 
then  made  in  bouillon,  and  after  2-t  hours  the  subculture  was  re- 
plated.  Subculturing  and  replating  were  then  repeated.  From  this 
last  plate  the  pure  culture  was  made  on  agar  for  study.  These  were 
not  studied  culturally,  as  a  rule,  for  some  weeks,  thus  allowing  time 
for  the  organism  to  eliminate  any  character  due  to  recent  environ- 
mental conditions  (l).a 

Morphology,    Staining    Properties,    and    Oxygen    Requirements,    with    Sug- 
gestions on  Variations. 

(a)  Size. — The  length  and  thickness  of  a  micro-organism  often 
varies  so  much  with  its  environmental  conditions  that  certain  re- 

«  Numbers  in  parentheses  refer  to  papers  in  the  bibliography  at  the  end  of 
Part  I  or  that  at  the  end  of  Part  II. 


10  THE    BACTERIA    OF    THE    APIAKY. 

corded  dimensions  should  always  be  accompanied  by  facts  concernino- 
the  medium,  age.  and  temperature  of  incubation.  The  measure- 
ments recorded  in  this  paper  were  all  taken  of  organisms  in  prepara- 
tions made  from  a  24-hour  agar  culture  stained  with  carbol-fuchsin. 
The  involution  forms  arc  not  reckoned  in  the  results. 

(b)  Spores. — The  presence  of  spores  was  determined  in  each  case 
by  staining  the  various  cultures  at  different  ages.  A  check  was  made 
on  their  presence  by  means  of  the  thermal  death  point. 

(c)  Flagella. — LoeihVr^  method,  as  modified  by  Johnson  and 
Mack,  was  used  for  staining  the  flagella  (2). 

(d)  Mot/lit;/. — Motility  may  be  present  in  cultures  when  first  iso- 
lated, but  after  artificial  cultivation  appear  to  be  entirely  lost.  The 
reverse  of  this  also  may  be  noted.  No  culture's  should  be  recorded 
asnonmotile  until  cultures  on  various  media  at  different  temperatures 
and  of  different  ages  -hall  have  been  studied.  Hanging-drop  prepar- 
tions  were  made  from  cultures  on  agar  and  bouillon,  both  incubated 
and  not  incubated,  and  on  gelatin. 

(c)  Staining  j>i<>j><  ,fi<  *. —  Basic  carbol-fuchsin  was  the  stain  u^cd 
almost  exclusively.  In  tin1  use  of  Gram's  staining  method,  carbolic 
gentian  violet  (5  per  cent  carbolic  acid  20  parts,  saturated  alcoholic 
solution  gential  violet  2  parts)  was  applied  to  a  cover-glass  prepara- 
tion from  a  2  l-hour  culture  on  agar  for  5  minutes,  placed  in  Lugol's 
solution  2  minute-,  and  placed,  without  rinsing,  in  95  per  cent  alcohol 
for  L5  minutes,  removed,  washt  in  water,  and  allowed  to  dry. 

(/')  Oxygen  requirements. — Determinations  were  made  by  ob- 
serving whether  a  growth  took  place  in  the  closed  or  open  arm  or 
both,  of  the  fermentation  tube  containing  glucose  bouillon. 

Media  Employed  and  Suggestions  as  to  the  Description  of  Cultures. 

(a)  Bouillon. — All  bouillon  used  was  made  from  beef  (meat  J 
part,  water  2  parts),  to  which  infusion  1  per  cent  Witte's  peptonum 
siccum  and  one-half  per  cent  sodium  chlorid  were  added.  The  re- 
action of  the  solution  was  then  determined  b}r  titrating,  and  made 
+1.5  to  phenolphthalein. 

In  describing  a  culture  growing  in  bouillon  as  a  medium,  there 
is  usually  a  more  extended  description  given  than  in  the  case  of 
sugar  and  sugar-free  bouillons,  since  cultures  in  these  media  do  not 
differ  materially  in  gross  appearance  from  those  observed  in  the 
plain  bouillon. 

(b)  Sugar-free  bouillon. — This  bouillon  is  made  free  from  sugar 
by  the  use  of  B.  coli  communis,  after  which  peptone  and  sodium 
chlorid  (NaCl)  were  added  as  in  bouillon. 

(c)  Sugar  bouillons. — Five  different  sugars — glucose,  lactose,  sac- 
charose, levulose,  and  maltose,  as  well  as  mannite — were  used  in  the 
study.    If  a  1-per-cent  solution  of  glucose  in  plain  bouillon  Avas  fer- 


MEDIA    EMPLOYED,  ETC:  11 

merited  with  the  production  of  gas,  fermentation  tubes  were  used 
for  all  the  sugars  and  mannite.  If  no  gas  was  formed  in  the  glucose, 
the  straight  tubes  were  inoculated.  The  sugars  and  mannite  were 
used  in  a  1-per-cent  solution  in  sugar-free  bouillon. 

(d)  Reaction  of  media. — The  reaction  of  cultures  is  determined 
as  it  appears  on  the  fifth  day  in  the  different  media,  unless  otherwise 
stated.  The  medium  in  the  open  arm  is  used  to  determine  the  re- 
action in  the  fermentation  tube.  Beginning  with  a  reaction  of  +1.5 
to  phenolphthalein,  or  slightly  alkaline  to  litmus,  the  detection  of  an 
increase  in  acidity  is  not  difficult.  But  inasmuch  as  the  production 
of  an  alkali  is  ver}?"  frequently  small  in  degree,  cultures  are  often  in 
this  paper  recorded  alkaline  in  reaction  when  probabty  the  reaction 
has  not  changed. 

(e)  Fermentation  with  the  production  of  gas. — Gas  may  be  formed 
in  such  small  quantities  as  not  to  be  observed  as  such,  but  to  be  en- 
tirely absorbed  by  the  medium.  Whenever  gas  formation  is  men- 
tioned as  a  character,  visible  gas  is  meant.  The  analysis  of  the  gas 
was  made  in  the  usual  manner  by  absorbing  a  portion  with  potassium 
hydrate  (KOH)  and  testing  the  remainder  with  the  flame.  The 
amount  absorbed  b}^  potassium  hydrate  (KOH)  is  referred  to  as 
carbon  dioxid  (C02)  and  the  remainder,  if  an  explosion  is  obtained, 
as  hydrogen  (H).  This  is,  naturally,  only  approximately  correct. 
Since  the  gas  formula  may  vary  from  day  to  day,  too  much  value 
must  not  be  given  to  the  exact  proportion.  It  is  well  to  observe 
whether  the  proportion  of  hydrogen  to  carbon  dioxid  is  greater  or 
less  than  1. 

(/)  Agar. — One  per  cent  agar  is  used.  The  description  of  the 
growth  on  this  medium  is  made  from  the  appearance  as  seen  on  the 
surface  of  an  agar  slant.  The  description  is  usually  very  brief,  since 
it  has,  as  a  rule,  little  differential  value. 

(g)  Acid  agar. — This  medium  is  made  acid  by  titrating  to  -\-3  to 
phenolphthalein.  The  absence  or  presence,  as  well  as  the  degree  of 
growth,  is  noted. 

(h)  Serum. — The  serum  used  is  taken  from  the  horse,  sterilized  at 
55°  C.  and  congealed  at  80°  C.  Deep  inoculations  are  made,  and  the 
surface  of  slanted  serum  is  also  inoculated.  The  degree  of  growth  is 
usually  noted.  Cultures  are  observed  for  6  weeks  to  2  months.  The 
presence  or  absence  of  liquefaction  is  the  chief  character  sought  for. 
Since  room  temperature  varies  so  greatly,  the  time  at  which  liquefac- 
tion begins  varies,  and  little  differential  value,  therefore,  can  be  given 
to  the  exact  time  of  this  phenomenon. 

(i)  Potato. — The  composition  of  potato  varies  so  markedly  that  a 
description  of  a  culture  on  this  medium  may  differ  materially  from 
that  which  is  observed  on  another  tube  of  the  same  medium.  It  is  the 
aim  to  omit  for  the  most  part  the  observed  variations  due  to  the 
composition  of  the  different  potatoes. 


12  THE    BACTERIA    OF    THE    APIARY. 

(j)  Potato  water. — To  potatoes  sliced  very  thin  is  added  an  equal 
amount  of -water  by  weight  and  the  mixture  is  then  boiled.  This  is 
strained  and,  distributed  in  straight  and  fermentation  tubes.  The 
reaction  of  the  solution  was  made  +1.5  to  phenolphthalein.  If  any 
of  the  micro-organisms  ferment  glucose  with  the  production  of  gas, 
fermentation  tubes  are  inoculated  to  test  the  fermentation  of  starch; 
if  not,  straight  tubes  are  inoculated. 

(/»•)  Milk. — If  a  micro-organism  breaks  up  glucose  with  the  forma- 
tion of  gas.  a  fermentation  tube  of  milk  is  inoculated  with  the 
culture;  if  not,  straight  tubes  are  used.  Separator  milk  is  used. 
The  coagulation  of  the  casein  with  or  without  liquefaction  is  the 
chief  character  noted.  Very  little  stress  is  laid  upon  the  time  ele- 
ment in  the  coagulation  of  the  casein  and  the  other  phenomena 
which  are  to  be  observed  in  milk.  Different  samples  of  milk  and 
the  different  environmental  conditions  are  factors  which  vary  the 
length  of  time  at  which  the  different   phenomena  appear. 

(/)  Litmus  milk. — The  reaction  a--  shown  by  the  litmus  and  the  dis- 
charging of  the  color  are  the  chief  points  observed. 

(m)  Gelatin. — The  color,  degree  of  growth,  the  presence  or 
absence  of  liquefaction,  and  the  form  of  liquefaction  are  the  chief 
points  observed.  The  culture-  are  kept  tinder  observation  2  months 
or  longer  and.  as  in  serum,  the  time  given  at  which  liquefaction  takes 
place  i-  only  approximate. 

(?i)  Indol. — The  culture-  are  allowed  to  grow  in  sugar-free  pep- 
tonized bouillon  for  3  to  5  days,  and  are  tested  with  potassium  nitrite 
(KN02)  and  sulfuric  acid  (H2S04)  after  the  ring  method.  Too 
much  stress  may  be  placed  upon  the  ability  of  an  organism  to  form 
indol.  This  character  has  been  shown  to  be  a  somewhat  transient 
one    (3). 

(o)  Reduction  of  nitrates  to  nitrites. — Cultures  are  cultivated  7 
•  lay-  in  a  solution  of  1  gram  of  YVitte's  peptonum  siccum  and  one- 
fifth  gram  of  sodium  nitrate  in  1,000  c.  c.  of  tap  water.  To  such  a 
(atlt ure  and  to  a  control  tube  are  added  a  mixture  of  naphthylamine 
and  sulfanilic  acid  (napthylamine,  1  part;  distilled  water,  1,000 
parts:  sulfanilic  acid,  one-half  gram,  dissolved  in  dilute  acetic  acid 
in  the  proportion  of  1  part  of  acid  to  16  parts  of  water).  If  nitrate 
is  reduced  to  nitrite,  a  pink  color  develops.  The  control  tube  should 
remain  clear,  or  slightly  pink — owing  to  the  absorption  of  a  trace  of 
nitrite  from  the  atmosphere. 

PART  I.  BACTERIA  OF  THE  NORMAL  APIARY. 

Before  studying  the  cause  of  a  disease  it  is  necessary  that  we 
know  what  bacteria  are  normally  present,  so  that  later,  in  studying 
diseased  conditions,  a  consideration  of  these  nonpathogenic  species 
may  be  eliminated.     In  view  of  this  necessity  a  bacteriological  study 


BACTERIA    FROM    THE    COMBS.  13 

of  the  hives,  combs,  honey,  pollen,  larvae,  and  adult  bees  was  begun, 
to  determine  the  bacteria  normally  present.  It  was  not  hoped  that 
all  the  species  isolated  could  be  easily  identified,  or  that  all  would 
merit  a  careful  description,  but  it  was  hoped  that  those  species  which 
seemed  to  be  localized  in  any  part  of  the  apiary,  or  upon  or  within  the 
bees,  might  be  studied  and  described  with  sufficient  care  to  guarantee 
their  identification  upon  being  isolated  again.  The  chance  of  varia- 
tion in  morphology,  pathogenesis,  and  cultural  characters  due  to 
environmental  conditions  to  which  these  micro-organisms  were  being 
subjected  at  the  time,  or  to  which  they  had  been  subjected  before 
isolation  or  study,  has  been  carefully  borne  in  mind. 

BACTERIA  FROM   THE   COMBS. 

One  might  naturally  suppose  that  very  many  species  of  bacteria 
would  be  present  on  combs,  since  these  are  exposed  more  or  less  to  the 
contaminating  influence  of  the  air.  The  reverse,  however,  seems  to 
be  true.  The  number  of  different  species  isolated  is  comparatively 
small.  Those  which  appear  most  often  are  described  below.  Some 
other  species  mentioned  in  this  paper  are  found  on  combs,  but  inas- 
much as  they  appear  most  frequently  from  other  sources  they  are 
described  there.  One  species  of  Saccharomyces  from  the  comb,  also, 
is  described  under  the  heading  "  Saccharomyces  and  fungi." 

Bacillus  A. 
(B.  mesentericus?) 

Occurrence. — Found  very  frequently  on  combs,  on  scrapings  from  hives,  and 
on  the  bodies  of  bees,  both  diseased  and  healthy. 

Gelatin  colonies. — Very  young  colonics  show  irregular  edges,  but  very  soon 
liquefaction  takes  place  and  the  colony  gives  rise  to  a  circular  liquefied  area, 
covered  with  a  gray  membrane,  which  later  turns  brown. 

Agar  colonies. — Superficial  colonies  present  a  very  irregular  margin  consist- 
ing of  outgrowths  taking  place  in  curves.  Deep  colonies  show  a  filamentous 
growth  having  a  moss-like  appearance. 

Morphology. — In  the  living  condition  the  bacilli  appear  clear  and  often  granu- 
lar, arranged  singly,  in  pairs,  and  in  chains.  The  flagella  are  distributed  over 
■•".he  body.  The  rods  measure  from  Sfi  to  4/a  in  length,  and  from  0.9/u  to  1.2,u 
in  thickness. 

Motility. — The  bacilli  are  only  moderately  motile. 

Spores. — Spores  are  formed  in  the  middle  of  the  rod. 

Gram's  stain. — The  bacilli  take  Gram's  stain. 

Oxygen  requirements. — Aerobic  and  facultatively  anaerobic. 

Bouillon. — Luxuriant  growth  in  24  hours,  with  cloudiness  of  medium  ;  a  gray 
flocculent  membrane  is  present.  Later,  the  membrane  sinks  and  the  medium 
clears,  leaving  a  heavy,  white,  flocculent  sediment,  with  a  growth  of  the  organ- 
isms adhering  to  the  glass  at  the  surface  of  the  medium.     Reaction  alkaline. 

Glucose. — Luxuriant  growth  takes  place  in  the  bulb,  with  a  moderate,  floccu- 
lent growth  in  closed  arm.     The  gradual  settling  of  the  organisms  causes  a 


14  THE    BACTEEIA    OF    THE    APIARY. 

heavy  white  sediment  to  form  in  the  bend  of  the  tube.  The  reaction  is  at  first 
slightly  acid,  but  subsequently  becomes  alkaline.     No  gas  is  formed. 

Lactose. — Reaction  alkaline. 

Saccharose.— ^Reaction  alkaline. 

Lcru1o.se — Reaction  acid. 

Maltose — Reaction  acid. 

Mannite. — Reaction  alkaline. 

Potato  water. — Reaction  alkaline. 

Agar  slant. — A  luxuriant  growth  takes  place  on  this  medium.  The  growth 
gradually  increases  to  a  moist,  glistening  one.  being  then  friable  and  of  a  grayish 
brown  color. 

Serum. — A  luxuriant,  brownish,  glistening,  friable  growth  spreads  over  the 
entire  surface.      No  liquefaction   is  observed. 

Potato. — An  abundant  fleshy  growth  of  a  brown  color  spreads  over  the  entire 
surface.     The  water  supports  a  heavy  growth.     The  potato  is  slightly  discolored. 

Milk. — Precipitation  takes  place  rapidly,  followed  by  a  gradual  digestion  of 
the  casein,  the  medium  changing  from  the  top  downward  to  a  translucent 
liquid,  becoming  ;it  last  semi-transparent  and  viscid. 

Lit  in  us  milk. — Precipitation  of  the  casein  takes  place  usually  within  24  hours, 
followed  by  a  gradual  peptonization.  Reduction  of  the  litmus  occurs  rapidly, 
'caving  the  medium  slightly  brown  :  later  the  blue  color  will  return  on  exposing 
the  milk  to  the  air  by  shaking.     Reaction  alkaline. 

('.(latin. — An  abundant  growth  takes  place  with  rapid,  infundibuliform  lique- 
faction. A  heavy,  white,  friable  membrane  is  formed  on  the  surface  of  the 
liquefied  medium.  A  flocculent  sediment  lies  at  the  bottom  of  the  clear  lique- 
fied portion. 

Acid  agar. — Growth  takes  place. 

Indol. — None  has  been  observed- 

Nitrate. — Reduction  to  nitrite  is  positive. 

Bacterium  acidiformans.     (Sternberg,  1892.) 

Occurrence,  isolated  from  the  scraping  of  propolis  and  wax  from  the  hives 
and  frames  of  healthy  colonies. 

Gelatin  colonies. — The  superficial  colonies  are  friable,  convex,  opaque,  and 
white  with  even  border:  when  magnified  they  are  finely  granular,  sometimes 
radiately  marked.  They  are  from  1  to  4  millimeters  in  diameter.  The  deep 
colonies  are  spherical  or  oblong  and  entire. 

Morphology. — When  taken  from  an  agar  slant  24  hours  old.  the  rods  are 
short,  with  rounded  ends,  singly  and  in  pairs.  Length  about  l.G/i,  thickness 
0.8/i.  They  stain  uniformly  with  carbol-fuchsin.  Flagella  are  apparently  ab- 
sent. 

Mot  Hit]/. — No  motility  has  been  observed  in  any  medium. 

Spores. — Spores  are  apparently  absent. 

drain's  stain. — The  bacteria  are  decolorized  by  Gram's  method. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon. — The  medium  becomes  slightly  clouded  with  a  feeble  ring  of  growth 
on  the  glass  at  the  surface  of  the  liquid.  A  moderate  amount  of  white  friable 
sediment  is  formed.     Reaction  alkaline. 

Glucose. — Uniformly  and  slightly  clouded.  No  gas  is  formed.  Reaction 
acid. 

Lactose. — Reaction  acid. 

Saccharose. — Reaction  alkaline. 

Levulose. — Reaction  acid. 


BACTERIA  FROM  POLLEN.  15 

Maltose. — Reaction  acid. 

Mannite. — Reaction  acid. 

Potato  water. — Reaction  acid. 

Agar  slant. — A  moderate,  gray,  glistening  growth,  confined  to  the  area  inocu- 
lated with  the  loop,  is  formed  on  the  inclined  surface. 

Serum. — A  feeble  gray  growth  is  formed  only  on  the  inoculated  surface.  No 
-liquefaction  takes  place. 

Potato. — A  gray  growth  covers  the  inoculated  surface. 

Milk. — Heat  causes  a  ready  coagulation  of  the  casein.     Reaction  acid. 

Litmus  milk. — Coagulation  of  casein  occurs  promptly  on  boiling  a  culture  2 
weeks  old.     Reaction  acid. 

Gelatin.— Growth  of  spherical  colonies  appears  along  the  line  of  inocula- 
tion, the  surface  growth  being  grayish  and  spreading  slowly.  No  liquefaction 
takes  place. 

Acid  agar. — Growth  takes  place. 

Indol. — A  trace  was  observed. 

Nitrate. — No  reduction  to  nitrite  could  be  observed. 

BACTERIA   FROM   POLLEN. 

As  in  the  case  of  the  examination  of  the  combs,  the  number  of  spe- 
cies of  bacteria  found  in  pollen  is  comparatively  small.  The  follow- 
ing are  often  found  to  be  present.  Other  species  have  been  isolated, 
but  their  distribution  in  the  pollen  is  not  at  all  constant. 

Bacillus  B. 

Occurrence. — Found  frequently  in  pollen  and  in  the  intestine  of  healthy 
honey  bees. 

Gelatin  colonics. — The  colonies  are  egg-yellow  with  even  border.  Liquefac- 
tion takes  place  slowly.  Surface  colonies  are  about  1.5  millimeters  in  diameter, 
have  coarsely  granular  center,  finely  granular  margin,  and  clear  and  sharply 
defined  border.     A  peculiar  toruloid  growth  is  often  observed. 

Morphology. — The  organisms  are  short  rods  with  rounded  ends,  which  stain 
uniformly  with  carbol-fuchsin,  and  are  1/*  to  2/j.  in  length.  Few  short  involu- 
tion forms  occur. 

Motility. — The  bacilli  are  actively  motile  in  young  cultures. 

Spores. — No  spores  have  been  observed. 

Gram's  stain. — The  bacilli  are  decolorized  by  Gram's  stain. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon. — This  medium  becomes  uniformly  clouded,  frequently  with  a  scanty, 
friable  membrane.  Sometimes  the  organisms  settle,  clearing  the  medium  and 
forming  a  viscid  sediment.  A  growth  of  the  culture  adheres  to  the  glass  at  the 
surface  of  the  liquid.  This,  together  with  the  membrane,  is  of  a  light  egg-yellow 
color,  which  deepens  somewhat  with  age.    Reaction  alkaline. 

Glucose. — At  first  both  arms  of  the  fermentation  tube  are  clouded  slightly,  and 
the  cloudiness  later  increases.  Sometimes  a  stronger  growth  occurs  in  the 
closed  arm  than  in  the  open  one.  Reaction  is  at  first  acid,  but  slowly  changes  to 
alkaline. 

Lactose. — Reaction  alkaline. 

Saccharose. — Reaction  alkaline. 

Levulose. — Reaction   alkaline. 

Maltose. — Reaction  slightly  acid. 
9583— No.   14— OG   m 3 


16  THE    BACTERIA    OF    THE    APIARY. 

Mannite. — Reaction  slightly  acid,  later  alkaline. 

Agar  slant. — A  moderate,  slightly  yellow,  nonviscid  glistening  growth  appears 
along  the  inoculated  surface.  This  growth  gradually  spreads  and  deepens  in 
color  to  an  egg-yellow. 

Potato. — A  moderate,  egg-yellow,  nonviscid.  glistening  growth  spreads  over 
the  entire  surface.     The  potato  is  slightly  discolored. 

Milk. — The  milk  is  covered  by  a  yellow  growth  of  the  culture,  resembling 
cream.     Coagulation  takes  place  on  boiling. 

Lit  hi  its  milk. — -Reaction  alkaline. 

Gelatin. — Growth  takes  place  along  the  line  of  inoculation.  Deep  in  the 
medium  the  colonies  arc  white  and  spherical:  the  surface  growth  is  yellow. 
After  :t  low  days  liquefaction  begins,  and  at  the  end  of  2  weeks  one-half  the 
tube  is  liquefied.  The  liquefaction  is  infundibuliform.  Liquefied  gelatin  is  sur- 
mounted by  a  friable,  egg-yellow  pellicle.  The  growth  in  the  liquefied  portion 
is  flocculent.  which,  on  settling,  forms  a  yellow  sediment  at  the  apex. 

Inclol. — None  could  be  observed. 

Nitrates. — No  reduction  to  nitrites  occurs. 

BACTERIA  IN  HONEY  AND  NORMAL  LARV^ffi. 

Comb  honey  from  a  Large  number  of  sources  has  been  examined 
and  found  to  be  quite  uniformly  sterile.  The  healthy  larvae  likewise 
are  usually  sterile. 

BACTERIA  UPON  THE  ADULT  BEES. 

On  the  external  part  of  the  bee  we  again  find  only  a  few  different 
species.  Bacillus  A,  described  as  found  upon  the  combs,  is  fre- 
quently isolated  from  the  bee.  Other  species  which  are  found  fre- 
quently are  described  below. 

Bacterium  cyaneus  (Micrococcus  cyaneus). 

Occurrence.— Isolated  from  the  body  of  ;i  healthy  honey  bee  and  from  pollen. 

Gelatin  colonies.— The  colonies  are  lemon-yellow,  with  entire  border,  growth 
taking  place  readily  on  this  medium.  The  superficial  colonies,  having  well- 
defined  border,  are  finely  granular,  and  liquefy  the  medium  within  3  to  G  days. 

Morphology. — Short  oval  rods  0.8a*  *<>  1-7/*  in  length,  0.7ft  to  0.8/t  in  thickness. 
Short  involution  forms  are  present.  The  rods  occur  singly,  paired,  and  in 
clumps.     No  flagella  have  been  demonstrated. 

Motility. — No  motion  has  been  demonstrated. 

Spores. — No  spores  have  been   demonstrated. 

Gram's  stain. — The  bacterium  takes  Gram's  stain. 

Oxygen  requirements. — Aerobic. 

Bouillon. — At  first  a  slight  cloudiness  appears,  the  medium  becoming  turbid 
in  old  cultures.  A  heavy  yellowish-white,  slightly  viscid  ring  forms  on  the 
tube  at  the  surface  of  the  medium.  The  sediment,  and  sometimes  the  medium, 
show  marked  viscidity.     Reaction  alkaline. 

Glucose. — The  growth  of  the  culture  is  confined  entirely  to  the  open  bulb,  in 
which  the  medium  becomes  turbid.     No  gas  is  formed.     Reaction  alkaline. 

Lactose. — Reaction  alkaline. 

Saccharose. — Reaction  alkaline. 

Levulose. — Reaction  alkaline, 


BACTEKIA    UPON    THE    ADULT    BEES.  17 

Maltose. — Reaction  alkaline. 

Mannite. — Reaction  alkaline. 

Potato  water. — Reaction  alkaline. 

Agar  slant. — On  the  surface  of  the  agar  there  takes  place  an  abundant  growth, 
which  is  confined  to  the  surface  inoculated  with  the  loop.  The  culture  is 
fleshy,  nonviscid,  and  lemon-yellow.  It  produces  a  soluble  pigment  that  dif- 
fuses thru  the  agar,  giving  it  a  dark-pink  color. 

Serum. — Luxuriant  growth  takes  place,  accompanied  by  liquefaction. 

Potato. — A  lemon-yellow,  fleshy,  glistening  growth  spreads  over  the  inclined 
surface  of  the  potato. 

Milk. — Precipitation  followed  by  slow  liquefaction  of  the  casein  occurs;  later 
the  medium  becomes  alkaline  and  very  viscid. 

Litmus  mill,-. — The  litmus  is  discharged  and  the  casein  is  liquefied.  Reaction 
alkaline. 

Gelatin. — Infundibuliform  liquefaction  soon  begins,  which  is  followed  by 
stratiform  liquefaction.     The  liquefied  gelatin  is  turbid  and  viscid. 

Acid  agar. — On  this  medium  a  moderate  lemon-yellow  growth  is  observed. 

Indol. — None  could  be  observed. 

Nitrates. — No  reduction  of  nitrates  could  be  observed. 

Micrococcus   C. 

Occurrence. — Isolated  from  the  body  of  a  healthy  honey  bee. 

Gelatin  colonics. — The  surface  colonies  are  round  and  slightly  yellow. 
Liquefaction  begins  in  from  2  to  4  days.  The  magnified  colonies  are  finely 
granular,  with  sharply  defined,  entire  border. 

Morphology. — Cocci,  about  0.8/x  in  diameter,  occur  in  pairs  and  in  small 
clusters. 

Motility. — Nonmotile. 

Spores. — Spores  are  apparently  absent. 

Gram's  stain. — The  coccus  takes  the  Gram's  stain. 

Oxygen  requirements. — Aerobic. 

Bouillon. — This  medium  becomes  uniformly  clouded  in  24  hours  after  in- 
oculation, growth  increases,  and  friable  sediment  forms.  The  liquid  clears 
somewhat  on  standing.  Reaction  at  first  slightly  acid;  later  returns  to 
neutral. 

Glucose. — The  medium  in  the  bulb  becomes  cloudy,  while  that  in  the  closed 
arm  remains  clear.  White  friable  sediment  forms  in  bend  of  tube.  Reaction 
acid.     No  gas  is  formed. 

Lactose. — Reaction  slowly  becomes  acid. 

Saccharose. — Reaction  acid. 

Levulose. — Reaction  acid. 

Maltose. — Reaction  acid. 

Mannite.— Reaction  acid. 

Potato  water. — Reaction  acid. 

Agar  slant. — A  grayish  white,  fleshy,  nonviscid,  glistening  growth  takes 
place  along  the  inoculated  surface.  It  does  not  spread,  and  retains  a  dis- 
tinct boundary. 

Serum. — A  spreading  growth  takes  place,  accompanied  by  liquefaction. 

Potato. — A  gray,  fleshy,  glistening,  nonviscid  growth  forms  over  the  entire 
cut  surface  of  the  potato.     The  potato  is  slightly  discolored. 

Milk. — This  medium  becomes  firmly  coagulated  and  later  the  casein  liquifies 
with  the  formation  of  a  milky  serum. 


18  THE    BACTERIA    OF    THE    APIARY. 

Lit  nuts  milk. — In  this  medium  Coagulation  takes  place,  accompanied  by 
reduction   of  the   litmus.      Reaction   slightly   acid. 

Gelatin. — After  a  day  or  two  infundibuliform  liquefaction  occurs,  being 
followed  by  stratiform  liquefaction;  the  liquefied  gelatin  is  turbid.  Growth 
below  this  portion  is  in  the  form  of  small  spherical  colonies. 

Acid  agar. — A  white,  fleshy.  Qonyiscid  growth  is  observed. 

Indol. — A  trace  was  observed. 

Vitrates. — Reduced  to  nitrites, 

BACTERIA  OF  THE  INTESTINE  OF  THE  HEALTHY  HONEY  BEE. 

A  great  many  investigations  have  been  made  in  recent  years  on  the 
bacteria  found  present  in  the  intestines  of  vertebrates  (  I.  5,  6,  7,  8, 
9),  and  striking  similarities  are  noticed  in  the  species  found  in  many 
of  them.  In  this  investigation  the  intestinal  contents  of  about  ir>0 
bees,  mostly  from  one  apiary,  have  been  studied  more  or  less  thoroly. 
Several  species  which  are  found  to  he  constant  in  many  of  the  verte- 
brates are  found  in  the  intestine  of  the  honey  bee.  Since  the  tem- 
perature of  the  bee  approximates  much  of  the  time,  especially  when 
in  the  hive,  that  of  the  warm-blooded  animals,  many  of  the  same 
species  of  bacteria  inhabit  the  intestine  of  this  insect  as  are  found 
thriving  in  the  same  locality  in  man  and  other  animals.  A  stained 
cover-glass  preparation  made  directly  from  a  healthy  adult  held  bee 
reveals,  almost  without  exception,  a  multitude  of  bacteria. 

In  a  study  of  the  bacterial  flora  stress  has  been  placed  upon  the 
different  species  which  were  found  to  be  more  or  less  constant,  rather 
than  upon  the  actual  number  of  bacteria  or  species  in  any  quantity 
of  material  from  a  single  bee.  From  the  observations  which  have 
been  made,  it  appears  that  the  number  of  species  in  any  individual 
is  comparatively  small,  but  the  number  of  bacteria  is  in  many  cases 
very  large.  Sometimes,  however,  the  piates  show  very  few  colonies, 
while  cover-glass  preparations  show  a  very  large  number  of  bacteria. 
These  organisms  are  probably  the  anaerobe,  which  is  quite  constant, 
as  shown  by  cultures  made  direct  from  the  intestine  into  glucose  agar 
(  Liborius's  method ). 

When  a  loopful  of  the  material  from  the  intestine  was  used  for  the 
inoculation,  the  following  data  give  the  approximate  findings: 

Bee  No.  1,300  to  -too  yellow  colonies,  probably  alike. 

Bee  No.  2.  a  few  colonies  of  fungi  only. 

Bee  No.  3,  500  colonies,  mostly  yeast. 

Bee  X<».    t.  loo  or  more  colon-like  colonies. 

Bee  No.  ">,  2,000  or  more,  mostly  yellow. 

Bee  No.  0.  20  or  more  colonies,  mostly  yeasts. 

Bee  No.  8,  400  or  more  yellow  colonies. 

Bee  No.  9,  30  yeasts  with  a  few  fungi. 

Bee  No.  10,  50  yeast  colonies  with  a  few  fungi. 

Bee  No.  1 1 .  no  growth. 

Bee  No.  12,  300  colonies,  slightly  yellow. 


BACTERIA    OF    THE    INTESTINE.  19 

Bee  No.  13,  2,000  or  more  gray  colonies. 

Bee  No.   14,  yeast  colonies   and  a  few  colonies  of  bacteria  showing  ground- 
glass  appearance.  % 
Bee  No.  15,  2,000  or  more  colon-like  colonies  (B.  cloacce). 

The  following  are  the  species  which  have  been  found  to  he  most 
constant.  The  reader  is  referred  also  to  the  description  of  the  yeast 
plant  found  very  frequently  in  the  intestine  of  the  normal  honey  bee, 
described  under  ,;  Saccharomyces  and  fungi." 

Bacterium  D. 

Occurrence. — Frequent  in  the  intestine  of  the  healthy  honey  bee. 

Agar  colony. — Deep  colonies  when  magnified  are  coarsely  granular,  showing  a 
dark  brown  center,  with  a  thin  and  ill-defined  border. 

Morphology. — A  preparation  made  from  a  young  culture  taken  from  a  glu- 
cose fermentation  tube  shows  rods  with  rounded  ends,  occurring  singly  and  in 
pairs,  staining  easily  and  uniformly  with  carbol-fuchsin.  and  measuring  0.7^  to 
1.5/4  in  length  and  0.5fi  to  0.7/t  in  thickness. 

Motility. — No  motility  could  be  observed. 

Spores. — No  spores  could  be  demonstrated  in  young  cultures.  In  old  cultures 
their  presence  is  questionable. 

Oxygen  requirements. — Strictly  anaerobic. 

Bouillon. — In  straight  tubes  no  growth  occurs. 

Glucose. — A  moderate  cloudiness  can  be  seen  in  the  closed  arm.  while  the 
open  bull)  remains  clear.     No  gas  is  produced.     Reaction  about  neutral. 

Glucose  agar  (Liborius's  method). — Growth  is  rather  slow.  After  3  days  a 
moderate  growth  may  be  observed:  later,  if  cultures  have  recently  been  iso- 
lated from  the  bee's  intestine,  the  growth  imparts  to  the  medium  a  diffused 
haziness  or  cloudiness.     After  many  generations  the  culture  loses  this  property. 

Glucose  gelatin  (Liborius's  method). — Very  slow  growth  occurs  in  the  depth 
of  the  medium.     No  liquefaction  takes  place. 

Bacillus  cloacae.  « 

Occurrence. — Found  in  the  intestine  of  a  large  number  of  healthy  honey  bees. 

Gelatin  colonics. — Superficial  colonies  are  thin  and  blue  to  gray  in  color;  deep 
colonies,  brown,  regular,  granular,  and  spherical  to  lenticular. 

Agar  colonics. — Superficial  colonies  are  partially  opaque,  brown,  finely  granu- 
lar, with  well-defined  margin;  deep  colonies  are  regular,  spherical,  or  lenticular, 
with  well-defined  margin. 

Morphology. — The  rods  from  24-hour  agar  cultures  have  rounded  ends,  vary- 
ing in  length  from  1/x  to  2m  and  in  width  from  0.7^  to  o.d/x.  They  are  usually 
found  singly  or  in  pairs.  Involution  forms  are  not  uncommon.  With  carbol- 
fuchsin  they  stain  uniformly.     This  species  possesses  a  few  peritrichic  flagella. 

Motility. — Active  motility  is  observed  in  young  cultures. 

Spores. — No  spores  are  formed. 

Gram's  stain. — The  bacillus  does  not  take  Gram's  stain. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon.— X  uniform  cloudiness  appears  in  21  hours.  Growth  continues  until 
the  medium  becomes  heavily  clouded,  followed  by  a  gradual  settling  of  many  of 
the  organisms,  forming  a  viscid  grayish-white  sediment.  A  gray  friable  mem- 
brane, which  adheres  to  the  sides  of  the  tube  at  the  surface  of  the  medium,  is 
sometimes  produced.     Upon  agitation  this  membrane  breaks  up  and  sinks  to  the 


20  THE    BACTERIA    OF    THE    APTARY. 

bottom,  leaving  a  gray  ring  of  the  growth  adhering  to  the  glass.  Reaction 
alkaline. 

Glucose.—  The  medium  in  the  bulb  becomes  turbid,  while  that  in  the  closed 
arm  is  "uniformly  cloudy.  A  heavy  grayish-white  sediment  is  formed.  The 
reaction  is  at  first  slightly  acid,  but  in  a  few  days  becomes  alkaline.  Abundant 
and  rapid  gas  formation  takes  place,  filling  usually  from  one-half  to  nine-tenths 
of  the  closed  arm.  The  ratio  of  hydrogen  to  carbon  dioxid  is  approximately 
1  to  2  ;    that  is.  the  ratio  of  hydrogen  to  carbon  dioxid  is  less  than  1. 

Lactose. — In  this  medium  gas  formation  takes  place  more  slowly  than  in 
glucose.  At  the  end  of  8  days  one-fourth  of  the  closed  arm  is  filled  with  gas. 
The  ratio  of  hydrogen  to  carbon  dioxid  is  greater  than  1.      Reaction  acid. 

Saccharose. — Gas  is  formed  abundantly  and  rapidly;  more  than  one-half  of 
the  tube  is  usually  filled  with  gas.  The  ratio  of  hydrogen  to  carbon  dioxid  is 
loss  than  1.     Reaction  alkaline. 

I.i  vulose. — A  rapid  fermentation  takes  place:  more  than  one-half  of  the  closed 
arm  is  filled  with  gas.  The  ratio  of  hydrogen  to  carbon  dioxid  is  approximately 
1  to  5;  that  is.  less  than  1.  A  slight  formation  of  acid  takes  place  at  first,  but 
the  reaction  rapidly  becomes  alkaline. 

Maltose. — Formation  of  iras  takes  place  with  the  result  that  at  the  end  of  5 
days  approximately  one-half  of  the  tube  is  tilled.  The  ratio  of  hydrogen  to 
carbon  dioxid  will  approximate  that  of  l  to  l.    Reaction  acid. 

Mannite. — Gas  is  formed  rapidly  and  abundantly;  at  the  end  of  5  days  the 
closed  arm  is  usually  much  more  than  half  tilled  with  the  gas.  The  reaction  is 
at  first  slightly  acid,  but  soon  becomes  alkaline.  The  ratio  of  hydrogen  to  car- 
bon  dioxid  is  approximately  1  to  2;  that  is.  less  than  1. 

Potato  water. — Gas  forms  rapidly  and  fills  half  the  closed  arm.  The  ratio  of 
hydrogen  to  carbon  dioxid  is  as  1   to  2  :   that  is.  less  than  1. 

Agar  slant. — A  moderate,  grayish-white,  glistening,  friable  growth  appears 
along  the  line  of  inoculation,  which  usually  spreads  to  the  sides  of  the  tube. 

Serum. — Moderate  gray  growth  appears,  which  is  confined  quite  closely  to  the 
line  of  inoculation.    Liquefaction  takes  place  slowly  after  .".  weeks. 

Potato. — A  moderate  amount  of  gray  fleshy  growth  covers  the  slope.  The 
potato  is  slightly  discolored. 

Milk. — Coagulation  takes  place  after  4  days'  growth.    Gas  is  formed. 

Litmus  mill,-. — A  marked  production  of  acid  takes  place,  followed  by  firm 
coagulation. 

Gelatin. — A  heavy  white  growth  takes  place  along  the  line  of  inoculation;  the 
surface  growth  is  Hat.  bluish-white,  and  spreads  with  an  uneven  margin.  Slow 
infundihuliform  liquefaction  takes  place  after  2  weeks. 

Acid  agar. — A  growth  takes  place. 

IndOl. — A  trace  is  sometimes  produced. 

Nitrates. — Reduction  to  nitrites  is  positive. 

B.  coli  communis. 

Occurrence. — Found  in  the  intestine  of  healthy  honey  bees. 

Gelatin  colonics. — The  superficial  colonies  are  blue,  lobate-lobulate,  and 
slightly  spreading;  when  magnified  they  are  brownish  yellow  in  the  center 
and  more  transparent  toward  the  margin;  the  deep  colonies  are  spherical  to 
lenticular  and  brownish  yellow,  with  well-defined  borders. 

Morphology. — The  short  rods  with  rounded  ends  measure  1.5/*  to  2/*  in  length 
and  0.7/*  to  0.8/t  in  thickness.  They  occur  singly  or  in  pairs,  stain  uniformly, 
and  are  motile  by  means  of  a  few  peritrichic  flagella. 


BACTERIA    OF    THE    INTESTINE.  21 

Motility. — The  bacilli  are  actively  motile  from  some  cultures. 

Spores. — No  spores  are  formed. 

Gram's  stain. — The  bacillus  is  decolorized  by  Gram's  method. 

Oxygen  requirements. — It  is  a  facultative  anaerobe.*     * 

Bouillon. — The  medium  becomes  uniformly  clouded  in  24  hours,  with  a  slight 
acid  reaction ;  the  medium  later  becomes  alkaline,  with  a  gray  and  friable 
sediment.  A  feeble  pellicle  is  formed  and  a  growth  of  the  organism  often 
adheres  to  the  glass  at  the  surface  of  the  liquid. 

Glucose. — Both  branches  of  the  fermentation  tube  become  clouded.  The 
sugar  splits  by  fermentation  into  gas  and  acid,  one-half  or  more  of  the  closed 
arm  being  filled.     The  ratio  of  hydrogen  to  carbon  dioxid  is  2  to  1. 

Lactose. — Gas  fills  one-fourth  of  the  closed  tube.     Reaction  acid. 

Saccharose. — Gas  fills  one-sixth  of  the  closed  tube.     Reaction  acid. 

Levulose. — Gas  fills  one-half  of  the  closed  tube.  The  value  of  hydrogen  to 
carbon  dioxid  is  2  to  1.     Reaction  acid. 

Maltose. — One-sixth  of  the  closed  arm  is  filled  with  gas.     Reaction  acid. 

Mannite. — One-half  of  the  closed  tube  is  filled  with  gas.     Reaction  acid. 

Potato  water. — Reaction  acid. 

Agar  slant. — A  moderate,  gray,  nonviscid,  spreading  growth  takes  place  on  the 
surface  of  the  inclined  agar. 

Serum. — A  gray,  glistening,  nonspreading  growth  is  observed  on  the  inclined 
serum.     No  liquefaction  takes  place. 

Potato. — A  moderate,  fleshy,  glistening  growth  spreads  over  the  inoculated 
surface.     Potato  slightly  discolored. 

Milk. — Coagulation  of  the  casein  takes  place  in  about  4  days.  A  small  quan- 
tity of  gas  is  produced. 

Litmus  milk. — Coagulation  occurs.     Reaction  strongly  acid. 

Gelatin. — A  moderate  growth  occurs  along  the  line  of  inoculation;  the  growth 
is  spreading  with  an  irregular  margin  on  the  surface.     No  liquefication  occurs. 

Acid  agar. — A  moderate  grayish  growth  occurs  on  surface. 

Indol. — A  trace  was  obtained  in  some  cultures. 

Nitrates. — Reduced  to  nitrites. 

B.  cholerae  suis. 

Occurrence. — Isolated  from  the  intestine  of  healthy  honey  bees. 

Gelatin  colonics. — Colonies  are  translucent  by  transmitted  light ;  bluish  to 
gray  by  reflected,  the  border  being  uneven  and  well  defined.  When  the  colonies 
are  magnified  they  appear  brownish  and  finely  granular. 

Morphology. — The  rods  are  short,  with  rounded  ends,  occurring  singly  and 
in  pairs,  and  staining  uniformly  with  carbol-fuchsin,  1  to  2.8/x  in  length,  and 
0.6/a  to  0.8/jl  in  thickness.     A  few  peritrichic  flagella  are  present. 

Motility. — Usually  only  a  few  are  motile  at  a  time  in  the  field,  and  these 
present  a  rapid  whirling  motion. 

Spores. — No  spores  are  formed. 

Gram's  stain. — The  bacteria  are  decolorized  by  Gram's  stain. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon. — A  uniform,  moderate  cloudiness  arises  in  this  medium  in  24 
hours ;  later  a  grayish-white  membrane  is  formed  which,  upon  shaking  the 
tube,  sinks  to  the  bottom,  forming  a  gray  sediment.  The  reaction  is  at  first 
slightly  acid,  but  later  becomes  alkaline. 

Glucose. — The  medium  becomes  clouded  in  both  arms  of  the  fermentation 
tube,  with  the  production  of  a  small  amount  of  gas.     Reaction  acid. 


22  THE     i;.\c  TKRTA    OF    THE    APIARY. 

Lactose. — Growth  takes  place  in  both  arms  of  the  tube,  hut  the  sugar  is  not 
split  into  either  acid  or  gas. 

•    Saccharose. — Growth  occurs  in  both  arms  of  the  tube,  neither  acid  nor  gas 
being  formed.    - 

Levulose. — Growth  takes  place  in  both  arms  with  the  production  of  gas  and 
acid;  one-third  of  the  closed  arm  is  filled.  The  ratio  of  hydrogen  to  carbon 
dioxid  is  about  :'>  to  1 — that  is.  greater  than  1. 

Maltose. — The  medium  in  both  arms  of  the  tube  becomes  clouded.  Fermenta- 
tion results  in  the  production  of  gas  sufficient  to  fill  about  one-fifth  of  the 
tube.  Only  a  small  portion  of  the  gas  is  absorbed  by  sodium  hydroxid.  leaving 
behind   an  explosive  gas. 

Mannite. — The  medium  in  both  brandies  of  the  tube  becomes  clouded:  gas 
is  not  formed.     Reaction  alkaline. 

Potato  water. — About  one-fifth  of  the  closed  arm  is  idled  with  gas.  Reaction 
acid. 

Agar  stunt. — A  moderate,  grayish-white,  glistening,  nonspreading  growth  is 
formed  along  the  surface  inoculated  with  the  loop. 

Serum.  A  moderate,  gray,  glistening,  nonspreading  growth  takes  place  on 
the  inclined  surface.     No  liquefaction  occurs. 

Potato. — A   feeble,  grayish  growth   is  observed.     The  potato  becomes  slightly 

discolored. 

Milk. — No  coagulation,  occurs,  and  no  gas  is  produced.     Reaction  alkaline. 
Lit  in  us  mill,-. —  The  medium  slowly  becomes  more  and  more  alkaline. 
Gelatin. — A    moderate,    white   growth    takes   place   along    the    line   of    inocula- 
tion.     On  the  surface  it   spreads  with  irregular  margin.      No  liquefaction  occurs. 
Acid  agar.— A  moderate  growth  appears. 
Indol. — Indol  is  produced. 
Nitrates. — Reduction  to  nitrites  »  ?). 

Bacillus  E. 

Occurrence. —  Isolated  from  the  intestine  of  healthy  honey  bees. 

Gelatin  colonies.-  The  colonies  are  lemon-yellow.  Surface  colonies  are  con- 
vex, smooth,  with  entire  margin:  when  magnified  they  are  finely  granular. 
Deep  colonies,  when  magnified,  are  lenticular,  finely  granular,  and  may  appeal 
dark  green.     Liquefaction  takes  place  slowly. 

Morphology. — The  rods  are  short,  with  rounded  ends,  and  usually  occur  singly. 
The  bacilli  are  1.5//  to  2//  in  length  and  0.7//  in  thickness.  This  species  pos- 
sesses a  few  peritrichic  flagella. 

1/'////////. — The  bacteria  are  actively  motile. 

Spores. — No  spores  are  present. 

Gram's  stain. — They  stain  with  drain's  stain. 

Oxygen  requirements. — Aerobic. 

Bouillon. — The  medium  becomes  uniformly  clouded  in  24  hours.  Later  a 
tough,  yellowish-white  membrane  is  formed,  which  sinks  upon  shaking.  The 
medium  is  very  viscid  in  old  cultures.     Reaction  alkaline. 

Glucose. — Growth  is  confined  to  the  open  bulb.  No  gas  formation  occurs. 
Reaction  slightly  acid. 

Lactose. — There  is  a  marked  mucous-like  appearance  in  the  medium.  Reac- 
tion alkaline. 

Saccharose. — Reaction  acid. 

Levulose. — Reaction  alkaline. 

Maltose. — Reaction  alkaline. 

Mannite. — Reaction  slightly  acid. 


BACTERIA    OF    THE    INTESTINE.  23 

Potato  water. — Reaction  alkaline. 

Agar  slant. — A  moderate,  yellowish-gray,  nonviscid  growth  takes  place  on  the 
surface. 

Serum. — A  strong  growth  takes  place  and  the  medium  is  liquefied. 

Potato. — A  yellowish-gray,  nonviscid  growth  is  observed  over  the  entire 
inclined  surface. 

Milk. — Precipitation  of  casein  takes  place  with  very  slight  digestion  (?). 

Litmus  milk. — Precipitation  of  the  casein  occurs.     Reaction  alkaline. 

Gelatin. — A  white  growth  forms  along  the  line  of  inoculation,  which  becomes 
slowly  liquefied  from  above. 

Acid  agar. — A  moderate,  slightly  yellow  growth  is  observed. 

Indol. — None  demonstrated. 

Nitrates. — No  reduction  to  nitrites  occurs. 

Bacillus  subgastricus. 

Occurrence. — Isolated  from  the  intestine  of  a  healthy  honey  bee. 

Gelatin  colony. — The  colon-like,  superficial  colonies  are  thin,  blue,  spreading, 
and  lobate-lobulate.  When  magnified  they  are  finely  granular,  with  brown 
center.     Deep  colonies  are  spherical  and  yellow. 

Morphology. — Short  rods,  singly  and  in  pairs,  are  from  1.5/a  to  2.5/x  long  and 
from  0.6/u,  to  0.8^  thick.     They  stain  uniformly  with  carbol-fuchsin. 

Motility. — Marked  whirling  motion  from  gelatin  cultures. 

Spores. — No  spores  could  be  demonstrated. 

Gram's  stain. — The  bacilli  are  decolorized  with  Gram's  stain. 

Oxygen  requirement*. — Facultatively  anaerobic. 

Bouillon. — This  medium  becomes  clouded  in  24  hours.  A  slight  band  of 
growth  is  formed  on  the  glass  at  the  surface  of  the  liquid.  Later  a  feeble 
pellicle  is  sometimes  formed.  Reaction  at  first  slightly  acid,  later  becomes 
alkaline. 

•  Glucose. — The  medium  in  both  branches  of  the  tube  becomes  clouded.  Gas 
is  readily  formed  until  about  one-fourth  of  the  closed  branch  is  filled.  The 
ratio  of  hydrogen  to  carbon  dioxid  is  2  to  i — that  is.  greater  than  1.  Reaction 
strongly  acid. 

Lactose. — Gas  formation  occurs.  About  one-sixth  of  the  tube  is  filled  with 
gas,  part  of  which  is  absorbed  by  sodium  hydroxid  and  another  part  is  explo- 
sive.    Reaction  acid. 

Saccharose. — This  sugar  is  fermented  to  the  point  of  formation  of  acid,  but 
no  gas  is  formed. 

Levulose. — This  sugar  splits  in  the  process  of  fermentation  to  form  acid 
and  gas,  the  gas  filling  about  one-sixth  of  the  tube.  A  portion  of  the  gas  is 
absorbed  by  sodium  hydroxid.  the  remainder  being  explosive. 

Maltose. — Fermentation  takes  place  with  the  formation  of  acid.  No  gas  is 
produced. 

Mannite. — One-fifth  of  the  closed  arm  is  filled  with  gas.  A  portion  of  the  gas 
is  absorbed  by  sodium  hydrbxid  and  a  portion  is  explosive.     Reaction  acid. 

Potato  water. — Reaction  alkaline. 

Agar  slant. — A  moderate,  translucent,  gray,  nonviscid  and  glistening  growth 
spreads  slowly  from  the  surface  inoculated  with  the  loop. 

Serum. — A  moderate,  glistening  growth  appears  along  the  surface  inoculated. 
No  liquefaction  occurs. 

Potato. — A  grayish  growth  takes  place  on  the  sloped  surface. 

Milk. — Firm  coagulation  of  the  milk  takes  place  with  the  formation  of  a 
small  amount  of  clear  serum.     A  small  amount  of  gas  is  produced. 
9583— No.  14— 06  m 4 


24  THE    BACTERIA    OF    THE    APIAEY. 

Litmus  milk. — Reaction  strongly  acid.     Coagulation  occurs  in  about  six  days. 

Gelatin. — White,  spherical  colonies  appear  along  the  line  of  inoculation.  The 
surface  growth  is  grayish  blue  and  spreading,  with  irregular  margin.  Slow 
liquefaction  takes  place,  beginning  usually  in  2  weeks. 

Acid  agar. — A  growth  takes  place. 

Indol. — None  could  be  demonstrated. 

Nitrates. — No  reduction  to  nitrites  occurs. 

Bacterium  mycoides. 

Occurrence. — Isolated  from  the  intestine  of  a  healthy  honey  bee. 

Gelatin  colonies. — A  rapid  growth  of  root-like  colonies  appears  in  24  hours. 
In  macroscopic  appearance  it  somewhat  resembles  cotton  fibers  ;  when  magni- 
fied these  appear  thick  and  somewhat  felted  in  the  center,  while  toward  the 
margin  they  are  beautifully  filamentous.  After  a  day  or  two  the  gelatin  begins 
to  liquefy. 

Morphology. — The  rods  are  large,  scarcely  rounded  at  the  ends,  and  frequently 
in  chains.  They  measure  from  2.5**  to  5.5/*  long  and  1.5a*  thick.  No  flagella 
have  been  demonstrated. 

Motility. — No  motility  could  be  demonstrated. 

spores. — Spores  a  it  present 

dram's  stain. — The  bacteria  are  not  decolorized  by  Gram's  stain. 

•Oxygen  requirement ts.— Facultatively  anaerobic. 

Bouillon. — A  decided  fleecy  growth  with  heavy,  cotton-like  sediment  occurs. 

(ili/cose. — No  gas  is  formed.     Reaction  acid. 

Lactose. — Reaction  acid. 

snrcharosc. — Reaction  acid. 

Lerulose. — Reaction  acid. 

Maltose. — Reaction  acid. 

Mannite. — Reaction  acid. 

Potato  water. — Reaction  alkaline. 

Agar  slant. — A  luxuriant  growth  that  appears  root-like  takes  place  on  this 
medium.  This  growth  tends  to  extend  into  the  agar,  which  causes  it  to  adhere 
to  the  medium. 

Serum. — A  luxuriant  growth  is  formed,  accompanied  by  liquefaction. 

Potato. — A  thick,  gray,  moist  growth  is  found,  the  potato  not  being  discolored. 

Milk. — Coagulation  occurs  promptly,  with  formation  of  a  clear  serum. 

Litmus  milk. — The  color  is  discharged  in  48  hours. 

Gelatin. — Hair-like  outgrowths  occur  along  the  line  of  inoculation.  Lique- 
faction begins  at  the  surface  and  proceeds  along  the  needle  tract.  In  a  few  days 
the  entire  medium  is  liquefied. 

Indol. — No  indol  is  produced. 

"Nitrates. — Reduction  to  nitrites  is  positive. 

Pseudomonas  fluorescens  liquefaciens. 

Occurrence. — Isolated  from  the  intestine  of  the  healthy  honey  bee. 

Gelatin  colonies. — Before  liquefaction,  the  superficial  colonies,  when  magni- 
fied, are  finely  granular,  with  regular  margin ;  deep  colonies  are  spherical, 
brown,  with  regular  margin.  Liquefaction  takes  place  rapidly.  The  surface 
of  liquefied  gelatin  is  covered  by  a  friable  membrane.  Later  the  liquefied  gela- 
tin takes  on  a  green  fluorescence. 

Morphology. — The  bacteria  are  short  rods,  varying  from  1/*  to  2/*  in  length 
and  from  0.5/*  to  0.7/*  in  thickness.  They  stain  uniformly  with  carbol-fuchsin 
and  are  motile  by  means  of  one  or  more  polar  flagella. 


SACCHAROMYCES    AND    FUNGI.  25 

Spores. — No  spores  could  be  demonstrated. 

Gram's  stain. — The  bacteria  do  not  take  Gram's  stain. 

Oxygen  requirements.— Aerobic 

Temperature  requirements. — Culture  must  be  grown  at  room  temperature. 

Bouillon. — The  medium  becomes  clouded  in  48  hours,  forming  a  moderately 
tough  pellicle.  A  greenish-yellow  fluorescence  begins  at  the  surface,  which 
gradually  increases  until  the  entire  medium  takes  on  that  appearance.  Reac- 
tion alkaline. 

Glucose. — A  cloudiness  is  formed  in  the  open  arm,  but  the  closed  arm  is  clear. 
Reaction  alkaline. 

Lactose. — Reaction  alkaline. 

Saccharose. — Reaction  alkaline. 

Levulose. — Reaction  alkaline. 

Maltose. — Reaction  alkaline. 

Mannite. — Reaction  alkaline. 

Agar  slant. — At  first  a  gray  friable  growth  is  formed  confined  to  the  surface 
inoculated,  which  later  takes  on  a  brown  hue.  Greenish-yellow  fluorescence  is 
observable  in  the  medium. 

Serum. — A  slow  liquefaction  occurs. 

Potato. — Very  scanty  growth  occurs  with  slight  discoloration. 

Milk. — Rapid  liquefaction  of  the  casein  takes  place. 

Litmus  milk. — Rapid  liquefaction  of  the  casein  takes  place.     Reaction  alkaline. 

Gelatin. — Infundibuliform  liquefaction  takes  place  rapidly. 

Acid  agar. — No  growth  occurs. 

IndoL — No  indol  observed. 

X  it  rates. — No  reduction  to  nitrites  occurs. 

SACCHAROMYCES   AND   FUNGI. 

The  first  yeast  plant  described  below  is  of  very  frequent  occurrence 
in  the  intestine  of  the  normal  bee.  Saccharomyces  roseus  can  be  iso- 
lated from  the  comb.  A  large  number  of  common  fungi  were  found 
in  the  flora  of  the  intestines  and  in  cultures  from  the  pollen  and 
combs. 

In  addition  to  the  above  the  third  Saccharomyces  here  described 
was  found  in  two  samples  of  brood  apparently  diseased,  which  could 
not  be  diagnosed  as  any  disease  commonly  known. 

Saccharomyces  F. 

Occurrence: — Very  common  in  the  intestine  of  healthy  honey  bees. 

Gelatin  colonies. — Colonies  form  slowly;  the  superficial  colonies  are  white, 
glistening,  convex,  capitate,  and  about  1  to  2  millimeters  in  diameter.  When 
magnified  they  are  finely  granular,  brownish  yellow,  with  entire  margin.  Deep 
colonies  are  finely  granular,  with  uniform  margin,  spherical  to  lenticular,  and 
brownish  green. 

Morphology. — The  cells  are  oval  and  on  agar  in  24  hours  attain  their  full 
size  of  4.5/i  in  length  and  3.5/u,  in  thickness.  They  stain  uniformly  with  carbol 
fuchsin. 

Motility. — The  yeast  is  not  motile. 

Gram's  stain. — The  cells  take  the  Gram's  stain. 

Oxygen  requirements. — Aerobic 


20  THE    BACTERIA    OF    THE    APIAEY. 

Bouillon. — This  medium  remains  clear,  with  the  formation  of  a  friahle  white 
sediment.     Reaction  neutral. 

Glucose. — The  closed  arm  remains  clear.    No  gas  is  formed.    Reaction  acid. 

Lactose. — Reaction  neutral. 

Saccharose. — Reaction  neutral. 

Levulose. — Reaction  neutral. 

Maltose. — Reaction  neutral. 

Mannite. — Reaction  neutral. 

Agar. — A  white,  nonspreading  growth  occurs. 

Serum. — White,  moderate,  nouviscid,  nonspreading  growth  occurs  along  the 
surface  inoculated.    No  liquefaction  takes  place. 

Potato  water. — Reaction  neutral. 

Potato. — Gray,  luxuriant,  fleshy  growth  occurs. 

Milk. — No  change  occurs. 

Litmus  milk.—  No  change  occurs. 

Gelatin. — A  moderate  growth  is  formed,  accompanied  by  no  liquefaction. 

Add  agar. — Moderate  growth  takes  place. 

Indol. — Negative. 

Nitrates. — Reduced  to  nitrites. 

Saccharomyces  roseus. 

Occurrence. — Isolated  from  comb  of  healthy  hive. 

Gelatin  colonics. — Superficial  colonies  are  pink,  convex,  capitate,  with  lobate- 
lobulate  margin  ;  when  magnified,  the  deep  colonics  are  irregular,  brownish- 
yellow,  and  finely  granular. 

Morphology. — This  cell  is  oval,  attaining  about  6.5/u  in  length  and  3.5/x  in 
thickness.     The  cells  stain  uniformly. 

Motility. — No  motility  occurs. 

Grant's  stain. — The  cells  arc  not  decolorized  by  Gram's  stain. 

Oxygen  requirements. — Aerobic 

Bouillon. — This  medium  remains  clear,  forming  a  pink,  friable  sediment.  A 
pink  band  forms  at  the  surface  of  the  medium  and  adheres  to  the  glass. 

Glucose. — The  closed  arm  remains  clear.     No  gas  is  formed.     Reaction  acid. 

Lactose. — Reaction  neutral. 

Saccharose. — Reaction  neutral. 

Levulose. — Reaction  slightly  acid. 

Maltose. — Reaction  slightly  acid. 

Mannite. — Reaction  neutral. 

Potato  water. — Reaction  acid. 

Glucose  agar. — Luxuriant,  red  growth  forms  on  the  surface. 

Serum. — A  pink,  fleshy,  nonspreading  growth  is  formed,  accompanied  by  no 
liquefaction. 

Potato. — A  thick,  nonspreading,  red  growth  occurs. 

Milk. — No  apparent  change  takes  place.     The  milk  coagulates  on  boiling. 

Litmus  milk. — Reaction  alkaline. 

Gelatin. — Moderate  pink  growth  is  formed,  accompanied  by  no  liquefaction. 

Acid  agar. — Slow  growth  occurs. 

Indol. — Negative. 

Nitrates. — Reduction  to  nitrites  is  positive. 

Saccharomyces  G. 

Occurrence. — Found  in  the  dead  larvaB  of  diseased  adult  bees. 

Morphology. — They    appear    in    hanging-drop   preparation    in    large   clusters, 


SACCHAROMYCES    AND    FUNGI.  27 

stain  uniformly  with  carbol-fucbsin  and  are  oval,  nearly  spherical,  attaining 
the  length  of  4.5/u  and  thickness  of  3.5/*. 

Gram's  stain. — The  cells  are  not  decolorized  by  Gram's  stain. 

Oxygen  requirements. — Aerobic. 

Bouillon. — A  slight,  friable,  white  sediment  is  formed,  with  a  clear  medium 
above.     Reaction  slightly  acid. 

Glucose. — The  medium  in  the  closed  arm  remains  practically  clear  and  about 
one-fifth  of  the  closed  arm  is  filled  with  gas.     Reaction  acid. 

Lactose. — Reaction  neutral. 

Saccharose. — Reaction  neutral. 

Levulose. — Reaction  slightly  acid. 

Maltose.— Reaction  slightly  acid. 

Mannite. — Reaction  neutral. 

Potato  water. — Reaction  acid. 

Agar. — A  moderate,  white  growth  is  formed. 

Serum. — Very  feeble  growth  occurs,  accompanied  by  no  liquefaction. 

Potato. — A  luxuriant,  moist,  white  growth  occurs. 

Milk. — No  appreciable  change  takes  place. 

Litmus  milk. — No  appreciable  change  takes  place. 

Gelatin. — A  moderate,  white  growth  occurs  along  needle  tract  and  on  the 
surface.     No  liquefaction  results. 

Acid  agar. — A  feeble  white  growth  occurs. 

Indol. — None  could  be  demonstrated. 

Nitrates. — No  reduction  to  nitrites  occurs. 

Glucose  agar. — A  thick,  white,  fleshy  growth  occurs. 


28 


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BIBLIOGKAPHY    TO    PART    I.  29 

SUMMARY  TO  PART  I. 

The  results  of  the  study  of  the  bacteria  found  normally  in  the 
apiary  may  be  briefly  summarized  as  follows : 

(1)  The  temperature  of  the  hive  approximates  that  of  warm- 
blooded animals. 

(2)  Upon  adult  bees  and  upon  the  comb  there  occurs  quite  con- 
stantly a  species  of  bacteria  which  we  refer  to  in  this  paper  as 
Bacillus  A.  and  which,  it  is  believed,  is  the  organism  that  some 
workers  have  confused  with  Bacillus  alvei,  the  cause  of  European 
foul  brood  (p.  33). 

(3)  There  occurs  very  constantly  in  the  pollen  and  intestine  of 
adult  bees  a  species  here  referred  to  as  Bacillus  B. 

(4)  From  the  combs  Bacterium  eyaneus,  Saccharomyces  roseus, 
and  a  Micrococcus  referred  to  here  as  Micrococcus  C,  have  been  iso- 
lated and  studied. 

(5)  Honey  from  a  healthy  hive  is,  as  a  rule,  sterile. 

(6)  The  normal  larva?  are,  as  a  rule,  sterile. 

(7)  There  is  an  anaerobe  found  quite  constantly  in  the  intestine  of 
the  healthy  honey  bee.    It  is  referred  to  jn  this  paper  as  Bacterium  D. 

(8)  From  the  intestine  there  have  been  isolated  and  studied  the 
following  micro-organisms:  Bacillus  cloaca?^  Bacillus  colt  communis. 
Bacillus  cholera^  suis,  Bacillus  subgastrieus*  Bacterium  ?nycoides, 
Pseudomonas  -fluoresceins  liquefaciens,  and  two  referred  to  as  Bacillus 
E,  and  Saccharomyces  F.  Others  less  frequently  present  have  been 
isolated,  but  not  studied. 

(9)  In  two  samples  of  brood  with  unknown  disease  there  was 
found  a  species  of  yeast  plant  here  referred  to  as  Saccharomyces  G. 

BIBLIOGRAPHY  TO  PART  I. 

1.  Fuller,  Geo.  W.,  and  Johnson,  Geo.  A.     On  the  Differentiation  and  Distribu- 

tion of  Water  Bacteria.     <Jour.  of  Exper.  Medicine,  Vol.  IV,  p.  609,  1899. 

2.  Johnson,  O.  P.,  and  Mack,  W.  B.     A  Modification  of  Existing  Methods  for 

Staining  Flagella.     < American  Medicine,  Vol.  VII,  p.  754,  1904. 

3.  Peckham,  Adelaide  W.     The  influence  of  environment  upon  the  biological  pro- 

cesses of  the  various  members  of  the  colon  group  of  bacilli.     <Jour.  of  Exp. 
Medicine,  Vol.  II,  No.  5,  p.  549,  1897. 

4.  Ford,  Wm.  W.     The  Classification  and  Distribution  of  the  Intestinal  Bacteria  in 

Man.     <Studies  from  the  Royal  Victoria  Hospital,  Montreal,  Vol.  I,  No.  5, 
1903. 

5.  King,  W.  E.     A  Study  of  the  Bacterial  Flora  of  the  Intestinal  Mucosa  and  Eye 

of  the  Common  Fowl.     <Thesis,  Cornell  University  Library,  1905. 

6.  Bullard,  M.  J.     A  Study  of  the  Bacterial  Flora  of  the  Intestinal  Mucosa  of  the 

Normal  Pvabbit.     <American  Medicine,  Vol.  IV,  No.  14,  pp.  546-548,  1902. 

7.  Dyar,  Harrison  G.,  and  Kieth,  Simon  C,  jr.     Notes  on  the  Normal  Intestinal 

Bacilli  of  the  Horse  and  of  other  Domesticated  Animals.     <Technological 
Quarterly,  Vol.  VI,  No.  3,  1893. 


30  THE    BACTERIA    OF    THE    APIARY. 

8.  Moore,  V.  A.,  and  Wright,  F.  R.     Observations  of  Bacillus  coli  communis 

from  certain  species  of  Domesticated  Animals.     <American  Medicine,  Vol. 
Ill,  No.  13,  p.  504,  1902. 

9.  Loeber,  E.     A  Bacteriological  Study  of  the  Intestine  of  the  Fish.     <Am.  Med., 

Vol.  VII,  No.  4,  p.  152,  1904. 

10.  Matzuschita,  T.     Bacteriologische  Diagnostik,  1902. 

11.  Chester,  F.  D.     A  Manual  of  Determinative  Bacteriology,  1901. 

PART  II.— THE  DISEASES  OF  BEES. 

The  bee  industry  in  this  country,  and  other  countries  as  well,  is 
suffering  large  losses  from  various  diseases  among  bees.  Those  which 
are  most  destructive  attack  the  brood  and  weaken  the  colony  by  kill- 
ing off  large  numbers  of  the  young  larvae  which  would  otherwise 
mature.  There  are  other  diseases  which  attack  the  adults  and  so 
decrease  the  strength  of  the  colony  in  that  way. 

In  order  to  combat  a  disease  to  the  best  advantage  it  is  clear  that 
its  cause  must  be  known,  as  well  as  the  means  by  which  the  infection 
is  transmitted  and  the  environmental  conditions  which  are  favorable 
for  the  breaking  out  of  an  epidemic.  The  brood  diseases  among  bees 
are  on  the  increase.  The  custom  of  selling  and  shipping  the  honey, 
which  is  now  carried  on  more  extensively  than  formerly,  the  manner 
in  which  the  products  of  the  apiary  are  handled,  and  the  absence  of 
a  general  knowledge  by  the  mass  of  bee  keepers  of  the  nature  of  the 
diseases  are  conditions  which  must  be  met  before  the  spread  of  these 
diseases  can  be  checked.  When  a  colony  is  diseased,  very  little  or  no 
profit  is  realized  from  it ;  consequently  the  wealth  and  comfort  of  a 
very  large  number  of  people  are  greatly  endangered  by  the  existence 
of  bee  diseases.  Thi>  suggests  the  importance,  from  an  economic 
standpoint,  of  a  thoro  knowledge  of  these  disorders. 

BRIEF    HISTORY. 

The  attention  of  investigators  has  been  attracted  by  these  diseased 
conditions,  not  only  from  the  economic  interests  attached  thereto, 
but  from  the  scientific  point  of  view  as  well.  The  writings  of  Aris- 
totle (12)  contain  an  account  of  certain  disorders  which  were  then 
prevalent  among  bees;  at  that  time  it  was  thought  that  the  blight  of 
flowers  bore  a  relation  to  bee  diseases.  In  1769  Schirach  (13)  gave 
the  name  foul  brood  to  a  diseased  condition  of  the  brood  of  bees; 
he  attributed  the  cause  to  (a)  unwholesome  food,  and  (b)  the  placing 
of  the  larvae  with  head  inward  in  the  cell.  Leuckhart  (14)  thought 
the  cause  to  be  a  fungus,  related  to  the  cause  (Panhistophyton  ova- 
tum)  of  the  disease  of  the  silkworm.  Muhlfeld  (15),  in  1868, 
thought  the  trouble  to  be  of  two  kinds — infectious  and  noninfec- 
tious— and  that  the  cause  of  the  infectious  one  is  the  larva  of  a  para- 
sitic fly  {Ichneumon  apium  melliftcarium)  feeding  upon  the  larvae  of 
the  bee.     In  1868  Preuss   (16)   exprest  the  view  that  the  cause  of 


BKIEF    HISTORY    OF    BEE    DISEASES.  31 

foul  brood  is  a  fermenting  fungus  belonging  to  the  genus  Cryptococ- 
cus.  Geilen  (17),  in  1868,  thought  that  when  bees  alight  on  the 
remains  of  animal  bodies  the  putrefying  matter  thus  carried  with 
them  may  cause  foul  brood.  The  fermentation  of  bee  bread  was 
thought  by  Lambrecht  (18)  to  be  a  sufficient  cause  of  the  disease; 
while  Hallier  (19)  thought  that  various  fungi  could  produce  the 
disorder.  On  the  contrary,  Cornallia  (20),  in  1870,  exprest  the 
opinion  that  a  fungus  (Cryptococcus  alvearis)  is  the  specific  cause  of 
the  trouble.  Fischer  (21),  in  1871,  supposed  that  a  predisposing 
factor  of  foul  brood  is  to  be  found  in  insufficient  nourishment.  In 
1874  Cohn  and  Eidem  received  from  Schonfeld  samples  of  foul  brood 
and,  upon  examination,  they  found  spores  and  rods.  In  1885  Chesh- 
ire and  Cheyne  (22)  determined  the  cause  and  named  the  germ 
Bacillus  alvei.  Dickel  (23)  claimed  that  a  number  of  different 
species  might  be  the  cause  of  foul  brood.  In  1900  Harrison  (24) 
writes  on  foul  brood  and  Bacillus  alvei,  its  cause.  Doctor  Lambotte 
(25),  in  1902,  made  some  interesting  studies  concerning  the  relation 
of  Bacillus  alvei  and  Bacillus  mesentericus  vulgatus. 

Since  so  many  conflicting  views  have  been  held  as  to  the  cause  of 
foul  brood,  one  might  conclude  that  the  term  "  foul  brood  "  has  been 
applied  incorrectly  to  a  number  of  different  disorders.  In  the  light 
of  more  recent  work  this  supposition  is  strengthened. 

In  June,  1902,  the  author,  under  the  direction  of  Dr.  Veranus  A. 
Moore,  began  an  investigation  of  bee  diseases,  especially  as  they  ex- 
isted in  Xew  York  State.  There  were  recognized  at  that  time  by 
bee  inspectors  of  that  State  a  number  of  distinct  diseases  which 
attacked  the  brood.  Those  which  caused  the  greatest  loss  to  the 
apiarists  were  known  to  the  bee  experts  as  "  black  brood,"  "  foul 
brood,"  and  "  pickle  brood."  The  results  of  the  investigations  of 
1902  (26),  1903  (27).  and  1904  (28)  on  these  disorders^  and  on  palsy 
or  paralysis,  are  embodied  in  the  following  pages. 

THE  TERM  "  FOUL  BROOD  "  AS  HITHERTO  APPLIED. 

In  the  discussion  of  foul  brood  of  bees  it  must  be  remembered  that 
until  recent  years  the  name  has  been  applied  to  what  is  now  known  to 
be  two  distinct  diseases. 

Schirach,  in  1769,  gave  the  name  foul  brood  to  a  diseased  condition 
in  the  brood  of  bees,  but  it  is  impossible  to  know  to  which  of  the  two 
he  referred.  It  may  be  that  both  diseases  existed  then  as  now  and 
that  he  did  not  observe  the  fact  that  the  two  were  different.  We 
have  reason  to  think  that  there  are,  at  the  present  time  in  Europe, 
two  distinct  diseases  to  which  the  name  foul  brood  is  being  applied. 
It  is  definitely  known  that  such  is  the  case  in  America. 

It  becomes  necessary,  then,  to  have  two  names  to  designate  these 


32  THE    BACTERIA    OF    THE    APIARY. 

two  diseased  conditions  in  the  brood  of  bees.  For  reasons  given  by 
Dr.  E.  F.  Phillips,  in  the  preface  to  this  paper,  it  has  been  considered 
advisable  to.  retain  the  name  foul  brood  and  to  use  a  qualifying  word 
to  distinguish  the  two  diseases.  "  European  foul  brood "  and 
'•American  foul  brood  "  are  the  names  by  which  these  two  diseased 
conditions  are  to  be  designated. 

In  1885  Cheyne  (22)  in  England  (Europe)  found  present  in  the 
decayed  larva'  suffering  from  a  diseased  condition  known  as  "foul 
brood"  a  new  bacillus,  which  he  named  Bacillus  alvei  ami  to  which 
he  ascribed  the  cause  of  the  disease.  The  diseased  condition  which 
contains  Bacillus  alvei  is  to  be  called  "  European  foul  brood,"  because 
this  fact  was  first  observed  by  an  investigator  working  in  Europe 
(England).  In  1903  (27)  the  author  observed  that  there  was  con- 
stantly present  in  the  other  diseased  condition  known  as  wt  foul  brood  " 
another  bacillus  which  was  new.  and  to  which  the  name  Bacillus 
/-'/■'•a  is  given.  In  view  of  the  fact  that  Bacillus  larva  was  con- 
stantly found  to  be  present  in  the  larva'  suffering  from  this  disorder 
in  the  brood  of  bee-,  by  investigations  carried  on  in  New  York  State 
(America)  (27)  (28),  this  diseased  condition  is  to  be  called  "Ameri- 
can foul  brood."  From  a  scientific  standpoint  this  choice  of  names 
for  two  distind  diseases  might  be  easily  criticized,  but  from  the 
standpoint  of  the  apiarist  the  selection  of  these  names  as  the  common 
one-  for  these  two  distinct  disorders  seemed  almost  necessary,  or  at 
least  advisable. 

EUROPEAN  FOUL  BROOD  (FOUL  BROOD  OF  CHEYNE). 

The  first  scientific  investigation  of  this  disease  bacteriologically 

was  performed  by  Cheyne  in  1885  (22).  At  this  time  he  isolated  a 
new  bacillus  from  the  dead  larva1.  It  was  described  by  him  and 
given  the  name  Bacillus  alvei  (literally,  hive  bacillus).  This  afforded, 
then,  a  means  for  a  positive  diagnosis  of  this  diseased  condition. 

Symptoms. 

The  symptoms  of  European  foul  brood,  as  given  by  Dr.  E.  F. 
Phillips  in  Circular  No.  79,  Bureau  of  Entomology,  are  as  follows: 

Adult  bees  in  infected  colonies  are  not  very  active,  but  do  succeed  in  cleaning 
out  some  of  the  dried  scales.  This  disease  attacks  larva4  earlier  than  does 
American  foul  brood,  and  a  comparatively  small  percentage  of  the  diseased 
brood  is  ever  capped;  the  diseased  larvae  which  are  gapped  over  have  sunken 
and  perforated  cappings.  The  larvae  when  first  attacked  show  a  small  yellow 
spot  on  the  body  near  the  head  and  move  uneasily  in  the  cell;  when  death 
occurs  they  turn  yellow,  then  brown,  and  finally  almost  black.  Decaying  larva? 
which  have  died  of  this  disease  do  not  usually  stretch  out  in  a  long  thread 
when  a  small  stick  is  inserted  and  slowly  removed  ;  occasionally  there  is  a  very 
slight  "  ropiness."  but  this  is  never  very  marked.  The  thoroly  dried  larva?  form 
irregular  scales  which  are  not  strongly  adherent  to  the  lower  side  wall  of  the 


CONFUSION    REGARDING    FOUL    BROOD    IN    AMERICA.  33 

cell.  There  is  very  little  odor  from  decaying  larvae  which  have  died  from 
this  disease,  and  when  an  odor  is  noticeable  it  is  not  the  "  glne  pot "  odor  of 
American  foul  brood,  but  more  nearly  resembles  that  of  soured  dead  brood. 
This  disease  attacks  drone  and  queen  larvae  very  soon  after  the  colony  is 
infected.  It  is,  as  a  rule,  much  more  infectious  than  American  foul  brood  and 
spreads  more  rapidly.  On  the  other  hand,  it  sometimes  happens  that  the 
disease  will  disappear  of  its  own  accord,  a  thing  which  the  author  never  knew 
to  occur  in  a  genuine  case  of  American  foul  brood.  European  foul  brood  is 
most  destructive  during  the  spring  and  early  summer,  often  almost  disap- 
pearing in  late  summer  and  autumn. 

Confusion  Regarding  Foul  Brood  in  America. 

Prof.  J.  J.  Mackenzie  in  1882  made  what  seems  to  have  been  a 
short  study  of  a  bee  disease  as  it  appeared  in  Ontario,  Canada,  which 
was  known  to  the  apiarists  of  that  Province  as  foul  brood.  He  says 
very  little  of  the  character  of  the  species  of  bacteria  with  which  he 
was  working,  but  he  supposed  that  they  were  Bacillus  alvei  of 
Cheyne.  The  author  has  examined  samples  of  brood  from  Ontario 
which  have  what,  in  the  opinion  of  bee  experts,  is  the  most  prevalent 
disease,  and  has  not  found  Bacillus  alvei  present  in  any  one.  The 
bacteriological  findings  and  the  experience  of  bee-disease  experts 
show  that  American  foul  brood  is  the  prevalent  disease  in  that  Prov- 
ince. As  the  bee  experts  see  the  disease  in  the  light  of  recent  studies, 
there  is  no  authentic  report  of  which  we  are  aware  that  European 
foul  brood  exists  in  Ontario.  We  can  safely  say,  then,  that  Bacillus 
alvei  can  not  be  isolated  from  larvae  taken  from  the  prevalent  disease 
in  the  above-named  Province.  No  difficulty  is  exprest  on  the  part 
of  Professor  Mackenzie  in  the  isolation  of  Bacillus  alvei  from  any 
sample.  The  author  is  inclined  to  think,  therefore,  that  this  investi- 
gator was  in  error  as  to  the  identity  of  his  culture,  and  therefore  his 
conclusion  can  have  little  weight. 

The  foul  brood  of  bees  received  some  attention  also  from  Prof. 
F.  C.  Harrison,  of  Ontario.  In  a  paper  of  some  length  he  gives  a 
description  of  a  species  of  bacteria  which  he  identified  as  Bacillus 
alvei.  The  description  which  he  gives  and  the  accompanying  photo- 
micrographs (another  plate  which  was  given  being  after  Cheyne 
and  correct  for  Bacillus  alvei)  might  easily  be  that  of  a  member  of 
a  group  represented  by  and  described  as  Bacillus  "J:"  in  Part  I  of 
this  paper.  He  also  says  that  he  has  isolated  Bacillus  alvei  from 
diseased  larvae  from  13  States  of  the  Union,  ranging  from  New 
York  to  California  and  from  Michigan  to  Florida.  European  foul 
brood  has  had  a  very  limited  geographical  distribution,  spreading 
only  recently  from  New  York  to  adjoining  States.  In  Professor 
Harrison's  work,  too,  there  seems  to  have  been  no  difficulty  in  iso- 
lating Bacillus  alvei  from  diseased  brood  diagnosed  by  bee  inspectors 


34  THE    BACTERIA    OF    THE    APIARY. 

as  foul  brood  thruout  the  United  States  and  Canada.  In  the  experi- 
ence of  the  author  it  has  not  been  possible  to  obtain  Bo  (ill  us  alvei 
from  diseased  brood  which  the  inspectors  in  most  of  the  States  and 
in  Canada  have  been  calling  foul  brood.  For  the  above  reasons  the 
author  believes  that  Harrison,  too,  has  made  a  serious  error  in  the 
identity  of  his  culture  and  therefore  was  not  working  with  Bacillus 
alvei  at  all.  The  author  considers  himself  unfortunate  in  that  he 
was  unable  to  obtain  a  culture  of  Bacillus  <tl 'vei  for  study  and  identi- 
fication from  Professor  Harrison. 

Dr.  William  1\.  Howard,  of  Fort  Worth,  Tex.,  also  studied  foul 
brood  somewhat,  and  gave  a  description  of  Bacillus  alvvi  as  he  found 
it.  From  his  description  and  from  the  fact  that  he,  too,  worked  with 
a  diseased  condition  which  does  not  contain  Bacillus  alvei,  and  ex- 
presl  no  difficulty  in  obtaining  his  cultures  from  any  samples,  the 
author  believes  that  this  investigator  made  an  error  in  the  identifica- 
tion of  the  culture  with  which  he  was  working. 

Some  writers — Cowan.  Bert  rand,  and  others — have  attempted  the 
positive  diagnosis  of  foul  brood  with  the  micro-cope  alone  from  a 
preparation  made  direct  from  the  dead  larva1.  If  the  reader  will 
remember  that  with  the  micro-cope  alone  it  would  be  impossible  to 
distinguish  between  Bacillus  larva  and  Bacillus  alvei,  the  verdict  of 
these  men  can  have  no  weight.  A-  shown  later  in  this  paper  under 
black  brood  (  pp.  \-\-\  \ ) .  the  Doctor  Howard,  of  Fort  Worth,  Tex.,  re- 
ferred to  above,  made  an  error  in  supposing  that  the  European  foul 
brood  was  a  new  disease  and  naming  it  "  New  York  bee  disease''  or 
"  black  brood." 

It  i>  very  unfortunate  for  the  apiarist  that  these  men  should  have 
fallen  into  error  as  to  the  identity  of  their  culture  with  Bacillus  alvei, 
as  it  has  caused  great  con  fusion  in  the  names  of  bee  diseases.  This 
confusion  in  the  identity  of  culture-  may  be  excused  to  a  certain  ex- 
tent by  the  fact  that  European  foul  brood  did  not  appear  in  this 
country,  or  at  leasl  did  not  attract  much  attention,  until  after  Mac- 
kenzie. Harrison,  and  William  K.  Howard  had  done  their  work  on 
foul  brood. 

The  Present  Investigation. 

TThen  the  author's  investigations  were  begun  in  1902  there  were 
two  especially  troublesome  diseases  in  this  country,  which  were  then 
known  to  the  bee  experts  as  "  black  brood  "  and  "  foul  brood." 

The  following  summary  and  table  shows  the  results  of  the  exami- 
nation of  a  number  of  samples  of  diseased  brood  from  different 
apiaries,  sent  by  the  New  York  State  bee  inspectors  during  the  sum- 
mer of  the  year  1902 : 


THE    PRESENT    INVESTIGATION    OF    EUROPEAN    FOUL    BROOD. 


35 


Table   shoivhig   the   results   of   examinations    of   European    foul    hrood.     {The 
samples  iccre  called  "black  brood'1''  by  the  apiarists  at  that  time.)% 


Brood  sent  by— 

Date. 

Bacteriological  findings. 

W.  D.  Wright 

Bacillus  alvei. 

W.  D.  Wright 

June  12 

Bacillus  alvei. 

N.  D.  West 

Bacillus  alvei. 

N.  D.  West 

June  12 

Bacillus  alvei. 

N.D.West 

June  12... 
June  12... 
June  12... 

Bacillus  alvei. 

N.  D.  West 

Bacillus  alvei. 

N.D.West 

Bacillus  alvei. 

N.  D.  West !  June  12 . 

N.  D.  West !  Aug.  5  . 

W.  D.  Wright I  Oct.  8. . . 


Bacillus 
Bacillus 
Bacillus 


alvei. 

alvei. 

dli-i  i. 


It  can  be  seen  clearly  from  the  above  table  that  the  diseased  condi- 
tion which  the  apiarists  were  calling  "  black  brood  "  is  really  the 
disease  "  foul  brood  "  of  Cheshire  and  Cheyne,  because  of  the  con- 
stant presence  of  Bacillus  alvei. 

The  work  upon  European  foul  brood  was  continued  during  the 
year  1903.  The  following  table  gives  the  results  of  the  examination 
of  specimens  received  during  that  year.  The  samples  were  taken 
from  different  apiaries. 

Table  (jiving  a  summary  of  the  examination  of  specimens  of  European  foul  brood 

("  black  brood  "). 


Brood  sent  by- 


Date. 


D.Wright May 

D.  Wright May 

D.  West Tune 

D.  West I une 

D.  West June 

D.  West June 

D.  West Tune 

D.  West July 

D.  West July 

D.  West July 

D.  West July 

D.  West !  July 

D.  West July 

D.West :  July 

D.West July 

I).  Wist J  July 


D.West. 
D.West. 
D.West. 
D.  West. 
D.  West. 
D.  West. 
D.West. 
D.  West. 
D.West. 
D.  West. 


July 

July 
July 
July 
July 

July 
July 

July 
Aug. 
Aug. 


Sources  of  brood  in  New  York. 


Columbia  County  . . . 

Albany  County.! 

Schoharie  County... 
Schoharie  County. .. 
Schoharie  County. .. 
Schoharie  County . . . 
Schoharie  County.. . 
Schoharie  County. . . 
Schoharie  County. .. 
Schoharie  County. .. 
Montgomery  County 
Schoharie  County . . . 
Schoharie  County. .. 
Schoharie  County. .. 
Schoharie  County. .. 
Montgomery  County 
Schoharie  County... 
Schoharie  County . . . 
Schoharie  County . . . 
Schoharie  County . . . 
Schoharie  County . . . 

Greene  County  . .' 

Albany  County 

Greene  County 

Greene  County 

Greene  County 


Bacteriological 
findings. 

Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvi  i. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 
Bacillus  alvei. 


The  above  table  showrs  that  Bacillus  alvei  was  present  in  each  speci- 
men of  European  foul  brood  received.  Frequently  pure  cultures 
of  this  species  were  obtained  from  dead  larvae,  but  with  it  sometimes 
Avere  associated  other  rod-shaped  bacteria  of  different  species. 

In  1904  the  work  upon  bee  diseases  was  confined  principally  to  the 
diagnosis  of  the  diseased  brood  sent  in  and  a  further  study  of  the 
organisms  found.     Bacillus  alvei  was  found  in  a  large  number  of 


36  THE    BACTERIA    OF    THE    APIARY. 

samples  received  from  New  York  State  and  in  some  received  from 
Pennsylvania. 

Bacillus  alvei. 

Occurrence. — This  bacillus  was  found  in  all  samples  of  European  foul  brood 
examined. 

Morphology. — The  bacillus  is  a  motile,  rod-shaped  organism,  occurring  singly 
and  in  pairs,  and  varying  when  taken  from  the  surface  of  agar  from  1.2u 
to  3.9/t  in  length,  and  from  0.5ft  to  0.7  m  in  width.  Involution  forms  are  some- 
times present  Spores  are  produced  and  occupy  an  intermediate  position  in 
the  organism.  They  are  oval  and  vary  from  1.5>  to  2/*  in  length  and  from 
0.7/*  to  1/u  in  breadth  :  they  exhibit  polar  germination.  The  few  flagella  are 
arranged  peritrichic.  , 

Oxygen  requirements. — This  bacillus  is  a  facultative  anaerobe  which  grows  at 
room  temperature,  but  better  at  37°  C. 

Bouillon. — The  medium  becomes  uniformly  clouded  in  24  hours;  later  it 
shows  a  tendency  to  clear  by  a  settling  of  the  organisms.  A  somewhat  viscid 
sediment  is  thus  formed  in  the  bottom  of  the  tube.  In  older  cultures  a 
slightly  gray  band  of  growth  adheres  to  the  glass  at  the  surface  of  the  me- 
dium. The  acidity  is  at  first  slightly  increased,  and  a  pellicle  is  sometimes 
formed. 

Glucose. — The  medium  in  both  branches  of  the  fermentation  tube  becomes 
uniformly  clouded.     (Jas  is  not  formed.      Reaction  acid. 

I. iiclo.se. — The  medium  becomes  uniformly  clouded  in  both  branches  of  the 
fermentation  tube,  hut  the  cloudiness  is  not  so  marked  as  when  glucose  is  used. 
The  acidity  is  slightly  increased,  as  shown  by  phenolphthalein.  No  gas  is 
formed. 

Saccharose. — The  bouillon  in  this  case  also  becomes  clouded  in  both  arms. 
A  heavier  growth  is  observed  than  when  lactose  is  used,  but  less  than  when 
glucose  is  used.     Acidity  is  slightly  increased.     Gas  is  not  formed. 

Agar  plates. — Small,  grayish,  circular  colonies  form  in  24  hours.  When  many 
are  on  the  plate,  they  do  not  exceed  2  millimeters  in  diameter.  Under  low 
magnification  they  appear  granular,  with  no  definite  margin.  When  fewer 
colonies  are  on  the  plate,  the  granular  center  of  the  colony  is  surrounded  by 
numerous  smaller  but  similar  growths.  The  organism  has  a  tendency  to  grow 
into  the  medium  rather  than  upon  the  surface.  Sometimes,  however,  when 
there  are  but  a  few  colonies  on  the  plate  a  thin,  transparent  growth  spreads 
rapidly  over  the  surface.     Later  it  takes  on  a  brown  tint. 

Agar  slant. — A  gray  layer  spreads  over  the  surface  in  24  hours,  which  later 
takes  on  a  slightly  brown  color.  A  strong,  slightly  viscid  growth  occurs  in  the 
condensation  water. 

Acid  agar. — Growth  takes  place  with  the  reactions  varying  from  neutral  to 
+3.5  to  phenolphthalein. 

Scrum. — A  slightly  raised  growth  which  is  confined  quite  closely  to  the  line 
of  inoculation  appears  on  the  surface  of  solidified  serum. 

Potato. — On  this  medium  the  bacillus  grows  rather  slowly  at  first,  but  after 
3  or  4  days  a  milky  growth  is  observed,  which  increases  until  a  luxuriant  growth 
is  formed,  which  varies  from  a  lemon-yellow  to  a  gray  color,  and  which  later 
becomes  tinted  with  brown. 

Milk. — Acidity  is  increased  after  inoculation.  Coagulation  usually  takes 
place  after  the  third  days 

Litmus  milk. — Much  of  the  blue  color  is  discharged,  leaving  the  coagulated 
milk  of  a  light  brown. 


INOCULATION    EXPERIMENTS    WITH    BACILLUS    ALVEI.  37 

Gelatin  colonies. — Gelatin  is  a  medium  in  which  it  develops  slowly.  The  col- 
ony becomes  very  irregular  in  outline,  owing  to  thread-like  outgrowths  which 
take  place  in  curves  from  its  border.  Growth  is  better  when  5  per  cent  glycerin 
is  added.  From  the  small,  white,  spherical  colonies  which  form  along  the  line 
of  puncture  gray,  thread-like  growths  shoot  out  thru  the  medium.  In  about  2 
months  the  gelatin  is  changed  to  a  thick  liquid,  holding  gray  floeculent  masses 
of  organisms  which  gradually  settle,  forming  a  strong,  slightly  viscid  sediment. 

Indol. — In  old  cultures  a  decided  indol  reaction  is  obtained. 

Power  to  resist  disinfectants. — Preliminary  observations  give  the  following 
results  :  The  spore  form  resists  drying  for  a  considerable  time.  Spores  which 
have  been  drying  for  1  year  germinate  promptly  when  introduced  into  bouillon. 
The  vegetative  form  :  One  per  cent  carbolic  acid  kills  in  10  minutes ;  3  per 
cent  carbolic  acid  kills  in  2  minutes ;  mercuric  chlorid  solution,  1  to  1,000. 
kills  in  1  minute ;   mercuric  chlorid  solution.  1  to  2,000.  kills  in  2  minutes. 

Spore  form. — Mercuric  chlorid.  1  to  1.000.  kills  in  30  minutes. 

Pathogenesis  in  vertebrates. — Inoculations  into  guinea  pigs  and  frogs  have 
not  proven  this  organism  to  be  pathogenic  to  these  animals. 

Inoculation  Experiments. 

That  part  of  the  investigation  which  involves  the  producing  of  the 
disease  experimentally  by  inoculating  with  pure  cultures  of  the 
organism  is  usually  the  most  difficult  one.  Very  rarely  indeed  is  one 
able  to  produce  the  disease  with  symptoms  closely  simulating  those 
found  in  nature.  The  experimental  production  of  a  disease  involves 
many  variable  factors,  such  as  attenuation  of  the  organism,  methods 
of  inoculation,  resistance  of  the  host,  and  the  immediate  environment. 

On  August  4.  1002,  we  inoculated  a  hive  containing  nothing  but 
healthy  brood,  free  from  bacteria,  by  feeding  with  sirup  (sugar  and 
water  in  equal  parts)  to  which  was  added  the  growth  from  the  sur- 
face of  the  plate  cultures  containing  spores  and  bouillon  cultures  of 
Bacillus  alvei.  Similar  feedings  were  given  to  these  bees  from  one 
to  three  times  a  week  until  September  28,  but  symptoms  of  foul 
brood  did  not  develop.  On  August  6  cultures  were  made  from  a 
few  of  the  hive  larvae.    They  were  found  to  contain  the  bacilli. 

Inoculation  experiments  were  again  made  in  1003.  Because  of  a 
failure  to  produce  a  diseased  condition  with  cultures  of  Bacillus  alvei 
in  the  experiment  of  1902.  the  variable  factors  above  mentioned  were 
carefully  considered  in  the  experiment  of  this  year.  The  inocula- 
tions were  made  when  climatic  conditions  were  such  as  seemed  to 
favor  the  ravages  of  the  disease  in  the  apiaries;  namely,  low  tem- 
perature, dampness,  and  cloudiness.  A  colony  of  black  bees  was 
used,  as  they  were  almost  universally  considered  more  susceptible. 
Cultures  of  BqcUIus  aire!  were  freshly  isolated  from  foul-brood 
specimens  and  kept  in  stock  on  bee-larvae  agar  (described  under 
American  foul  brood,  pp.  41—12).  All  cultures  were  incubated  at  34° 
C,  which  temperature  is  observed  to  be  slightly  below  that  of  the 
hive.    The  spore  form  of  Bacilli's  alvei  was  used. 

Inoculations  were  made  in  different  ways.     A  diseased  condition 


38  THE    BACTERIA    OF    THE    APIARY. 

appeared  in  the  hive  when  the  following  method  was  used :  The  agar 
from  plates  on  which  the  culture  was  grown  was  finely  crusht  and 
mixt  with  sterile  sirup.  A  jelly  glass,  in  the  lid  of  which  holes  had 
been  punctured,  was  filled  and  inverted  on  strips  of  wood  inside  the 
hive.  In  this  way  the  bees  take  up  the  culture  with  the  sirup  as 
rapidly  as  it  flows  out  of  the  glasses.  A  colony  having  brood  free 
from  Bacillus  alvei  was  fed  in  the  above  manner  on  August  8,  with 
repeated  feedings  on  the  9th,  10th,  12th,  13th,  15th,  and  17th.  On 
the  12th  Bacillus  alvei  was  found  in  the  living  larvae  and  on  the  17th 
many  larvae  were  dead  under  cappings  and  some  were  dead  which 
were  not  capped;  all  were  soft  and  of  a  dull  color.  Many  of  the 
capped  cells  containing  dead  larva1  had  their  capping  freshly  punc- 
tured. Badllu8  alvei  was  usually  obtained  from  these  larva4  in  pure 
cultures.  In  no  cell  examined  where  the  cell  capping  was  punctured 
did  Ave  find  gas-producing  organisms;  this  fact  would  suggest  the 
conclusion  thai  these  punctures  which  are  found  in  the  capping  in 
foul  brood  are  made  by  the  bees  and  not  by  gas-producing  organ- 
isms. During  this  series  of  inoculations  the  day-  were  quite  cool 
and  sometimes  cloudy  and  damp.  On  the  20th  of  August  the  tem- 
perature was  much  higher,  the  bees  were  more  active,  and  much  of 
the  dead  brood  had  been  cleaned  out  by  the  bees.  On  the  22d  no 
dead  brood  was  noticed  by  casually  looking  over  the  brood  nest.  On 
the  24th  of  the  same  month  a  careful  searoh  was  made  by  uncapping 
all  the  cells  of  one  brood  frame,  and  12  decaying  larva1  of  a  brown 
color  were  found.  At  this  time  the  larvae  were  not  viscid.  All  the 
remaining  dead  brood  had  evidently  been  cleaned  out  by  the  bees. 
A  condition  similar  to  this,  where  only  a  few  scattered  about  in  the 
brood  nests  contain  dead  larva1,  occurs  sometimes  in  affected  apiaries. 
Two  other  colonies  which  were  near  by  but  not  inoculated  gave  no 
signs  of  disease. 

Mr.  X.  1).  West  reports  that  the  climatic  conditions  seem  to  have 
something  to  do  with  the  extent  of  the  ravages  of  European  foul 
brood;  since  the  disease  is  much  more  destructive  in  cool,  damp 
weather.  This  seems  to  be  a  very  plausible  idea.  The  larva1  at  such 
times  may  receive  more  infected  food  than  when  fresh  is  being 
rapidly  gathered;  the  resistance  of  the  body  of  the  larva)  to  the 
growth  of  Bacillus  alvei  is  at  such  times  much  lessened;  and  the 
adult  bees  being  less  active,  the  dead  larvae  are  not  cleaned  out  of  the 
combs  so  rapidly.  The  results  of  the  experimental  work  seem  to 
confirm  this  theory. 

Distribution  of  Bacillus  alvei  in  Infected  Hives. 

In  order  to  combat  this  disease  it  is  well  to  know  where  these  patho- 
genic bacteria  may  be  found.  The  following  is  a  summary  of  the 
results  of  the  investigation  along  this  line : 


EXPEKIMENTS    WITH    FOKMALDEHYDE    GAS.  39 

1.  The  greatest  number  of  infecting  germs  are  found  in  the  bodies 
of  dead  larva?. 

2.  The  pollen  stored  in  the  cells  of  the  foul-brood  combs  contains 
many  of  these  infecting  organisms. 

3.  The  honey  stored  in  brood  combs  infected  with  this  disease  has 
been  found  to  contain  Bacillus  alvei  in  small  numbers. 

4.  The  surface  of  the  combs,  frames,  and  hives  may  be  contami- 
nated. 

5.  The  wings,  legs,  head,  thorax,  abdomen,  and  intestinal  contents 
of  adult  bees  are  found  to  be  contaminated  with  Bacillus  alvei. 

6.  Cheshire  (29),  Mackenzie  (30),  and  others  have  found  Bacillus 
alvei  in  the  ovary  of  the  queen.  This  has  suggested  a  means  of  in- 
fection. From  a  bacteriological  examination  of  queens  from  three 
badly  infected  hives  we  were  able  to  isolate  Bacillus  alvei  in  small 
numbers  in  two  cases.  Since  a  very  large  number  of  this  species  of 
bacteria  may  be  found  in  the  intestinal  tract  and  upon  all  parts  of 
the  body,  it  is  very  probable  that  such  findings  are  the  results  of  con- 
tamination in  making  cultures  and  have  no  special  significance. 

Experiments  with  Formaldehyde  Gas. 

Within  the  last  few  years  several  articles  have  appeared  in  the  bee 
journals  entertaining  great  hopes  that  a  cure  for  foul  brood  has  been 
found  in  the  use  of  formaldehyde  gas.  The  methods  described  for  its 
use  have  been  tested  by  the  apiarists  and  bee  experts  in  New  York 
State,  with  the  result  that  the  disease  sometimes  breaks  out  anew  in 
colonies  so  treated. 

In  order  to  test  the  value  of  formaldehyde  gas  as  a  disinfectant 
when  used  in  foul-brood  combs  a  number  of  experiments  were  made 
in  the  laboratory.  A  common  frame  hive  Avas  first  used,  in  which 
were  placed  specimens  of  foul  brood.  The  hive  was  charged  with 
gas  by  heating  formalin  in  a  closed  vessel  which  was  in  communica- 
tion with  the  hive;  15  c.  c.  was  used  each  time  and  evaporated  to 
dryness.  The  charging  of  the  hive  with  gas  was  repeated  in  this 
way  at  the  end  of  2,  4,  G,  and  20  hours.  Before  each  charging  and 
at  the  end  of  24  hours  after  the  first  application  of  gas,  cultures  were 
made.  Of  all  the  tubes  inoculated  90  per  cent  showed  Bacillus  alvei 
to  be  present.  There  was  no  decrease  in  the  number  of  tubes  in 
which  B  dell  his  alvei  appeared  following  the  several  applications  of 
formaldehyde  gas. 

The  examination  of  specimens  of  foul  brood  which  had  been  treated 
with  the  gas  by  an  apiarist  gave  the  following  results : 

Thirty  tubes  which  were  inoculated  from  larva?,  capped  and  un- 
capped, showed  the  presence  of  Bacillus  alvei  in  21. 

Thirty  tubes  which  were  inoculated  with  pollen  in  cells  gave 
Bacillus  alvei  in  28. 


40  THE    BACTERIA    OF    THE    AJPIAKY. 

Four  series  of  agar  plates  showed  apparently  no  diminution  in 

the  number  of  bacteria  present. 

Further  experiments  were  made  by  using  Novy's  anaerobic  jar 
(a  very  tight  chamber)  as  a  chamber  in  which  to  put  the  diseased 
brood  combs  and  cultures.  This  vessel  will  retain  the  gas  much  more 
perfectly  than  the  devices  made  for  practical  use  in  the  apiary. 
Treatment  of  brood  in  this  jar  by  recharging  with  the  gas  resulted 
usually  in  complete  disinfection  after  2  days.  Agar  plates  con- 
taining spores  and  cheese  cloth  on  which  cultures  were  spread  and 
dried  were  disinfected  after  a  short  length  of  time  by  the  applica- 
tion of  formaldehyde  gas. 

From  the  experiments  made  the  conclusion  can  be  drawn  that 
formaldehyde  gas  is  a  good  disinfectant,  but  that  it  penetrates  very 
slowly  and  that  24  hours'  application  of  the  gas  to  the  combs,  as 
usually  applied,  is  not  sufficient  to  kill  all  the  spores  in  the  deca}^ed 
larvae  (27). 

AMERICAN  FOUL  BROOD. 

The  diseased  condition  which  we  shall  call  American  foul  brood 
and  the  micro-organism  found  constantly  present  in  the  diseased  and 
dead  larvae,  which  we  shall  call  Bacillus  larvce,  were,  for  convenience, 
referred  to.  respectively,  as  "X  Brood"  and  Bacillus  "X"  in  a 
former  report  (ii~).  This  disease  has  been  called  "foul  brood"  by 
many  bee  keepers  in  this  country  and  in  other  countries  as  well.  It 
is  the  diseased  condition  with  which  Mackenzie.  Harrison,  and 
William  R.  Howard  were  working  largely,  if  not  altogether,  in  their 
investigation-  of  foul  brood.  The  disorder  is,  as  a  rule  dreaded  less 
than  European  foul  brood  by  the  apiarist,  yet  in  the  aggregate  the 
bee  industry  suffers  enormous  losses  Prom  the  trouble.  The  general 
character  of  the  diseased  brood  i-  so  much  like  that  of  foul  brood 
that  the  two  may  be  easily  confused  by  those  unfamiliar  with  the 
variety  of  appearances  which  one  buds  in  each  disease  and  a  few 
characters  which  are  differential.  Therefore  it  is  not  strange  that 
the  mistaken  diagnosis  should  be  made  from  the  symptoms  mani- 
fested by  these  two  diseases.  When,  however.  European  foul  brood 
and  American  foul  brood  are  subjected  to  a  bacteriological  exami- 
nation, the  diagnosis  is  easy.  Experts  when  comparing  specimens 
of  the   two   diseased  conditions   are   able   to  see  a  difference  in  the 

gross  appearance. 

Symptoms. 

The  symptoms  are  given  by  Dr.  E.  E.  Phillips  in  Circular  No.  TO. 
Bureau  of  Entomology,  as  follow- : 

The  adult  bee*  of  an  infected  colony  are  usually  rather  inactive  and  do  little 
toward  cleaning  out  infected  material.  When  the  larvae  are  first  affected  they 
turn  to  a  light  chocolate  color,  and  in  the  advanced  stages  of  decay  tbey  become 


THE    PKESENT    INVESTIGATION    OF    AMERICAN    FOUL    BROOD.        41 

darker,  resembling  roasted  coffee  in  color.  Usually  the  larvae  are  attacked  at 
about  the  time  of  capping,  and  most  of  the  cells  containing  infected  larva?  are 
capped.  As  decay  proceeds  these  cappings  become  sunken  and  perforated,  and, 
as  the  healthy  brood  emerges,  the  comb  shows  the  scattered  cells  containing 
larvae  which  have  died  of  disease  still  capped.  The  most  noticeable  charac- 
teristic of  this  infection  is  the  fact  that  when  a  small  stick  is  inserted  in  a 
larva  which  has  died  of  the  disease,  and  slowly  removed,  the  broken-down 
tissues  adhere  to  it  and  will  often  stretch  out  for  several  inches  before  break- 
ing. When  the  larva  dries  it  forms  a  tightly  adhering  scale  of  very  dark 
brown  color,  which  can  best  be  observed  when  the  comb  is  held  so  that  a  bright 
light  strikes  the  lower  side  wall.  Decaying  larvae  which  have  died  of  this  disease 
have  a  very  characteristic  odor,  which  resembles  a  poor  quality  of  glue.  This 
disease  seldom  attacks  drone  or  queen  larva1.  It  appears  to  be  much  more 
virulent  in  the  western  part  of  the  United  States  than  in  the  East. 

A  microscopic  preparation  from  the  diseased,  but  not  dead  larvae, 
or  from  larvae  recently  dead,  at  first  shows  a  few  comparatively  long 
slender  rods;  later  these  increase  rapidly  in  number,  and  spores  also 
are  seen.  In  the  later  stages  of  decay  in  the  ropy  mass  and  the  dried 
scales  spores  only  are  found;  these  occur  in  very  large  numbers. 
When  this  investigation  Avas  begun,  in  1902,  it  was  observed  (26) 
that  in  the  dried  dead  larvae  there  are  very  large  numbers  of  spores, 
but  these,  when  inoculated  into  the  media  commonly  used  in  the 
laboratory,  fail  to  grow.  The  cultures  were  sterile,  except  for  an  oc- 
casional contamination. 

The  Present  Investigation. 

The  following  samples  from  different  sources  were  examined  in 
1902: 

Results  of  cram ination  of  specimens  of  American  foul  brood  diagnosed  by  the 
experts  at  that  time  simply  as  "foul  brood.' 


Brood  sent  by — 

Date. 

Source. 

Bacteriological 
findings. 

Charles  Stewart 

June  12 

Sept.  19 

Oct.  19 

Nov.  11 

No  growth. 

W.  D.  Wright 

W.  D.  Wright 

cilli. 

No  growth. 

No  growth;  4  sam- 
ples. 

W.  D.  Wright 

Inasmuch  as  Bacillus  flZm'.was  absent,  it  is  evident  that  this  condi- 
tion is  not  European  foul  brood  (2G). 

In  1903  the  investigations  were  continued.  Several  media  were 
devised  in  which  it  was  hoped  that  it  would  be  possible  to  obtain  a 
germination  of  the  spores  which  were  observed  the  year  before  and 
which  failed  to  grow7  on  our  ordinary  media.  The  one  which  proved 
successful  was  prepared  as  follows :  Larvae  are  picked  from  the  brood 
combs  of  a  number  of  frames  of  healthy  brood  and  a  bouillon  (bee- 
larvae  bouillon)  is  made  from  them  following  the  same  directions  as 
when  bouillon  is  made  from  meat.     Our  first  growth  from  these 


42  THE    BACTERIA    OF    THE    APIARY. 

spores  was  secured  in  an  agar  (bee-larvae  agar)  made  from  this  special 
bouillon  when  liborius's  method  for  cultivating  anaerobes  was  used. 

The  technique  for  making  cultures  successfully  from  the  diseased 
material  is  not  difficult  if  the  following  method  is  used:  Place  a 
loopful  of  the  decayed  tissue  of  the  larva'  into  a  tube  of  bouillon; 
heat  to  G5°  C.  for  10  minutes  to  kill  any  vegetative  forms  which  might 
be  present;  incubate  for  1*_>  hours,  and  heat  again  to  65°  C.  for  10 
minutes.  This  is  usually  sufficient,  but  it  may  be  necessary  to  repeat 
the  same  process.  Liquefied  bee-larva4  agar  in  a  test  tube  is  then  in- 
oculated and  incubated.  The  successive  heating  will  destroy  the  veg- 
etative stage  of  any  -pore-producing  species  which  is  common  about 
the  apiary,  e.  </..  members  of  the  group  represented  by  Bacillus  A,  as 
described  on  pp.  13-14  of  this  paper.  Agar  slant  and  bouillon,  when 
inoculated  from  this  source,  remain  sterile;  but  when  bee-larva?  agar 
Is  used  a  -low  but  abundant  growth  takes  place.  Under  certain  con- 
dition^ the  growth  appears  very  near  or  at  the  surface  when  cultures 
are  made  in  the  above  manner.  A  surface  growth  can  be  obtained 
after  a  few  generations  by  reinoculating  slant  agar  of  this  same 
medium. 

The  above  method  was  used  successfully  in  diagnosing  the  follow- 
ing samples  from  different  apiaries: 

Results  of  examination  of  specimens  of  American  foul  brood,  formerly  c<iih<l 

simply  "  foul  brood." 


Brood  sent  by— 

Date. 

Source. 

Bacteriological 
findings. 

W    I>   Wright... 

Oct    19,1902 
NOV.    11,1902 

Nov.  11. 1902 
Julv  24,1908 
Aim.     3,1903 
An-.    3,1903 
Aug.     3,1903 

Canada 

W.  D.  Wright 

W.  1).  Wrighl 

C.  II.  W.  Weber 

N.  I).  West 

X.   D.  West 

Wisconsin 

Wisconsin 

Ohio 

Bacillus  larvae. 
Bacillus  larvss. 

Broome  County,  X.  v 

Broome  County,  X.  Y 

Chenango  County,  X.  Y 

BacUlui  larvss. 

X.  I).  West 

The  results  of  these  examinations  show  that  Bacillus  larvce  was 
present  in  all  the  specimens  examined,  which  suggests  that  it  very 
probably  figures  as  an  etiological  factor  in  this  disease.  Other  bac- 
teria of  different  species  are  occasionally  found  associated  with  this 
bacillus. 

Bacillus  larvae. 

Occurrence. — Constantly  present  in  diseased  brood  from  colonies  affected  with 
American  foul  brood. 

Gelatin. — There  is  no  growth. 

Morphology. — It  is  a  slender  rod.  having  a  tendency  to  form  in  chains.  This 
is  especially  true  when  grown  in  bee-larva1  bouillon. 

Motility. — The  bacillus  is  rather  sluggishly  motile. 

Spores. — Spore  formation  takes  place.  This  can  be  observed  best  in  the  dif- 
ferent stages  of  the  disease  and  decay  of  the  larvae. 

Oxygen  requirements. — When  Liborius's  method  is  used,  the  best  growth 
usually  appears  near  to  but  not  on  the  surface.  After  a  few  generations  a 
surface  growth  may  be  obtained. 


THE    SO-CALLED  " BLACK   BROOD."  43 

Bouillon. — There  is  no  growth. 

Glucose  bouillon. — There  is  no  growth. 

Lactose. — There  is  no  growth. 

Saccharose. — There  is  no  growth. 

Agar  plate. — There  is  no  growth. 

Bee-larva1  agar. — The  inoculations  must  be  made  with  the  medium  liquefied. 
The  growth  takes  place  near  tp  but  rarely  on  the  surface.  Cultures  must 
pass  thru  a  few  generations  before  a  satisfactory  surface  growth  can  be 
secured. 

Bee-larnv  agar  slant. — On  the  surface  of  this  medium  a  thin,  gray,  nonviscid 
growth  takes  place. 

Glucose  agar. — Slight  growth  has  been  observed  in  the  medium.  No  gas  is 
produced. 

Potato. — There  is  no  growth. 

Milk. — There  is  no  growth. 

Litmus  milk. — There  is  no  growth. 

Fermentation. — In  bee-larva?  bouillon  no  gas  is  produced. 

Indol. — There  is  no  growth  in  sugar-free  bouillon. 

THE  SO-CALLED  "  PICKLE  BROOD." 

The  name  "  pickle  brood  "  was  given  by  Dr.  William  R.  Howard,  of 
Fort  Worth,  Tex.,  to  a  disorder  found  in  the  brood  of  bees.  He 
stated  that  the  cause  of  the  disease  was  a  specific  fungus  which  he 
called  Aspergillus  polli?iis.  His  results  have  not  been  confirmed  by 
other  investigators. 

The  bee  keepers  are  sustaining  a  loss  from  a  diseased  condition  in 
their  apiaries  which  they  are  diagnosing  as  "  pickle  brood."  The 
larva1  usually  die  late  in  the  larval  stage.  Most  of  them  are  found 
on  end  in  the  cell,  the  head  frequently  blackened  and  the  body  of  a 
watery,  granular  consistency. 

The  following  table  gives  a  summary  of  the  results  of  an  examina- 
tion of  specimens  received  labeled  "  pickle  brood :  " 

Results  of  examination   of  specimens  of  so-called   "pickle  brood." 


Brood  sent  by—  Date. 


Bacteriological  findings. 


W.  D.  Wright June  17,1902 Two  unidentified  micrococci. 

W.  D.  Wright July  31, 1902 No  growth. 

W.  D.  Wright Aug.  4, 1902 ;  No  growth. 

M.  Stevens Aug.  20, 1902 Unidentified  bacilli. 

W.  D.  Wright Sept.  2, 1902 j  Unidentified  bacilli. 

W.  D.  Wright June  24, 1903 Unidentified  bacilli  and  yeast. 

N.  D.  West Aug.  5..1903 No  growth. 

M.  Stevens Aug.  20, 1903 No  growth. 

The  results  of  the  examinations  show  that  Aspergillus  pollinis  was 
not  found..  Further  investigations  must  be  made  before  any  conclu- 
sion can  be  drawn  as  to  the  real  cause  of  this  trouble. 

THE  SO-CALLED  "  BLACK  BROOD." 

In  1890  some  specimens  of  diseased  brood  were  sent  from  Xew 
York  State  to  Dr.  William  R.  Howard,  of  Fort  Worth,  Tex.,  and 
unfortunately,  after  a  short  and  inadequate  study  of  the  disease,  he 


44  THE    BACTERIA    OF    THE    APIARY. 

reported  it  to  be  a  new  disease  and  called  it  *;  New  York  bee  disease  " 
or  "  black  brood."  He  described  as  its  cause  a  species  of  bacteria 
which  he  called  Bacillus  millii  (31). 

In  our  investigations  of  this  diseased  condition,  which  have  covered 
five  years,  we  have  not  found  an  organism  corresponding  to  Bacillus 
millii  in  any  of  the  specimens  that  we  have  received;  but  we  have 
found  Bacillus  alvei,  the  supposed  cause  of  foul  brood,  to  be  present 
constantly  in  samples  of  brood  which  the  bee  experts  of  New  York 
State  say  are  samples  of  the  same  diseased  condition  as  that  received 
by  Howard. 

From  this  Ave  conclude  that  the  diseased  brood  that  has  received 
the  name  of  "New  York  bee  disease"  or  "black  brood"  is  really 
genuine  European  foul  brood. 

PALSY  OR  PARALYSIS. 

The  dfsease  known  to  the  apiarists  as  palsy  or  paralysis  attacks 
the  aduH  bees.  The  name  is  suggestive  of  the  symptoms  manifested 
by  the  diseased  bees.  A  number  of  bees  affected  were  received  from 
Messrs.  W.  I).  Wright  and  Charles  Stewart,  taken  from  apiaries  in 
New  York  State  In  L903  bacteriological  examinations  were  made  of 
a  number  of  bees  so  affected.  Several  species  of  bacteria  were  isolated 
and  sonic  experimental  inoculations  made,  but  no  conclusions  could  be 
drawn  from  the  results  obtained  a-  to  the  cause  of  the  disorder. 

From  a  study  of  the  normal  flora  of  the  bee  it  was  soon  found 
that  we  had  here  quite  a  number  of  species  of  bacteria  present. 
'J'his  fact  stimulated  a  study  of  the  normal  flora,  the  results  of  which 
are  recorded  in  Part  I.  From  this  point  the  work  can  be  carried 
on  with  the  hope  that,  if  the  disease  has  a  bacterium  as  an  etiological 
factor,  it  may  be  found.  It  is  believed  by  some  bee  keepers  that 
Bacillus  gaytoni  of  Cheshire  is  the  cause  of  paralysis,  but  this  is  not 
claimed  by  Cheshire,  and  the  belief  is  not  grounded  on  bacteriological 
findings. 

SUMMARY  TO  PART  II. 

Following  is  a  brief  summary  of  the  results  of  the  present  investi- 
gation of  bee  diseases: 

(1)  There  are  a  number  of  diseased  conditions  which  affect  the 
apiary. 

(2)  The  disease  which  seems  to  cause  the  most  rapid  loss  to  the 
apiarist  i>  European  foul  brood,  in  which  is  found  Bacillus  alvei — 
firsl  isolated,  studied,  and  named  by  Cheshire  and  Cheyne  in  1885. 

(3)  The  distribution  of  Bacillus  alvei  in  the  infected  hive  is  as 
follows : 

(a)  The  greatest  number  of  infecting  germs  are  found  in  the 
bodies  of  dead  larvae. 

(b)  The  pollen  stored  in  the  cells  of  the  foul-brood  combs  contains 
many"  of  these  infecting  organisms. 


CONCLUSIONS.  45 

.    (c)   The  honey  stored  in  brood  combs  infected  with  this  disease 
has  been  found  to  contain  a  few  bacilli  of  this  species. 

(d)  The  surface  of  combs,  frames,  and  hives  may  be  contaminated. 

(e)  The  wings,  head,  legs,  thorax,  abdomen,  and  intestinal  con- 
tents of  adult  bees  were  found  to  be  contaminated  with  Bacillus  alvei. 

(/)  Bacillus  alvei  may  appear  in  cultures  made  from  the  ovar}^  of 
queens  from  European  foul-brood  colonies,  but  the  presence  of  this 
species  suggests  contamination  from  the  body  of  the  queen  while  the 
cultures  are  being  made  and  has  no  special  significance. 

(4)  The  disease  which  seems  to  be  most  widespread  in  the  United 
States  we  have  called  American  foul  brood,  and  the  organism  which 
has  been  found  constantly  present  in  the  disease  we  have  called 
Bacillus  larva-.  This  disorder  was  thought  by  many  in  this  country 
and  other  countries  as  well  to  be  the  foul  brood  described  by  Cheshire 
and  Cheyne,  but  such  is  not  the  case. 

(5)  From  the  nature  of  American  foul  brood  it  is  thought  that  the 
organism  has  a  similar  distribution  to  that  of  Bacillus  alvei. 

(6)  It  appears  that  European  foul  brood  was  erroneously  called 
"  Xew  York  bee  disease  "  or  "  black  brood  "  by  Dr.  Wm.  R.  Howard 
in  1900. 

(7)  There  is  a  diseased  condition  affecting  the  brood  of  bees  which 
is  being  called  by  the  bee  keepers  "  pickle  brood."  Xo  conclusion  can 
be  drawn  from  the  investigation  so  far  as  to  the  cause  of  the  disease. 

(8)  Aspergillus  pollinis,  ascribed  by  Dr.  William  E.  Howard  as 
the  cause  of  pickle  brood,  has  not  been  found  in  this  investigation 
and  is  not  believed  by  the  author  to  have  any  etiological  relation  to 
the  so-called  "  pickle  brood." 

(9)  Palsy  or  paralysis  is  a  diseased  condition  of  the  adult  bees. 
No  conclusion  can  yet  be  drawn  as  to  its  cause. 

(10)  Formaldehyde  gas  as  ordinarily  used  in  the  apiaries  is  insuffi- 
cient to  insure  complete  disinfection. 

CONCLUSIONS. 

In  a  paragraph  the  author  wishes,  if  possible,  to  present  the  status 
of  the  bee  diseases  in  this  country.  It  should  be  remembered,  firstly, 
that  "  black  brood  "  can  now  be  dropt  from  our  vocabulary,  and 
probably  does  not  exist ;  secondly,  that  the  term  "  foul  brood  "  was 
being  applied  to  two  distinct  diseases.  One  of  these  diseases  we  now 
refer  to  as  European  foul  brood,  because  it  first  received  a  scientific 
study  from  a  European  investigator.  We  refer  to  the  other  disease 
as  American  foul  brood,  because  it  was  first  studied  scientifically  in 
America.  There  is  one  more  disorder  in  the  brood  of  bees  which  has 
attracted  considerable  attention — the  so-called  "  pickle  brood." 
There  are,  then,  these  three  principal  diseases:  European  foul  brood, 
American  foul  brood,  and  the  so-called  "  pickle  brood." 


46  THE    BACTERIA    OF    THE    APIARY. 

BIBLIOGRAPHY  TO  PART  II. 

12.  Aristoteles.     <Historia  Animalium,  Book  IX,  Ch.  27. 

13.  Schleach,     <Histoire  des  Abeilles,  Ch.  3,  p.  56,  1769. 

14.  Leuckhart.     <Binen-zeitung.     Eichstadt,  p.  232,  1860. 

15.  Muhlfbld.     <Bienen-zeitung.     Eichstadt,  p.  232,  1868. 

16.  Preobs.     <Bienen-zeitung,  p.  95,  1868. 

17.  Geilen.     <Bienen-zeitung.  Nos.  21  and  22,  1868. 

18.  Lambrecht.     <Bienen-zeitung,  No.  2,  1870. 

19.  Hallier.     <Bienen-zeitung,  No.  2,  1870. 

20.  Corn  a  i. li  a.     <Bienen-zeitung,  No.  5,  1870. 

21.  Fischer.     <Bienen-zeitung,  p.  105,  1871. 

22.  Cheshire  and  Cheyne.     The  pathogenic  history  and  history  under  cultivation 

of  a  new  bacillus  (B.  alvei)  the  cause  of  a  disease  <>f  hive  beea  hitherto  known 
as  foul  brood.     <Jour.  Roy.  Mic.  Soc,  Vol.  V.,  p.  581,  1885. 

23.  Dickel.     <  Bienen-zeitung,  p.  124,  1888. 

24.  Harrison,  F.  C.     The  foul  brood  of  bees.     <Bulletin  No.  112,  Ontario  Agric. 

College.     Also  in  Centralblatt  fur  Bakteriologie,  Parasitenknnde  und  Infek- 
tionskrankheiten,  Zweite  Abtheilung,  VI  Band,  1900. 

25.  Lambotte.     Recherches  sur  le  Microbe  de  la  "Loque."  maladie  des  abeilles. 

<Annales  de  l'lnstitut  Pasteur,  Vol.  XVI,  p.  694,  1902. 

26.  Moore,  V.  A.,  and  White,  <;.  Franklin.     A  report  on  the  investigation  of  an 

infectious  bee  disease.     <Ne\v  York  State  department   of  agriculture,  Jan., 
1903. 

27.  White,  <i.  Franklin.     A  report  of  the  further  investigation  of  bee  diseases  of 

the  State  affecting  the  apiaries  of  the  State  of  New  York.     •    New  York  State 
department  of  agriculture,  Jan.,  1904. 

28.  White,  G.  Franklin.     A  report  of  the  work  on  bee  diseases  for  1904.     <New 

York  State  Department  of  Agriculture,  Jan.,  1905. 

29.  Cheshire.     <  Bees  and  beekeeping.     Vol.  II,  London.     1885. 

30.  Mackenzie.     Ontario  Agricultural  College  Report,  1893. 

31.  How  ai:d,  Wm.   R.     New  York  Bee  Disease,  or  Black  Brood.        Gleanings  in 

Bee  Culture,  Feb.  15,  1900. 

32.  Benton.     •  [Bulletin  of  Apiculture,  No.  4,  1*86. 

33.  Smith,  W.  G.     <British  Bee  Journal.  Vol.  XIV,  p.  1225,  1*86. 

34.  Jones,  S.  A.     Foul  Brood,  its  management  and  cure.     <Beeton,  Canada,  1886. 

35.  McLean.     ^Department  of  Agriculture  Report.     Washington,  p.  584,  1886. 

36.  Ward,  F.  F.     <British  Bee  Journal,  p.  396,  1887. 

37.  Schredter.     <Bienen-zeitung,  1887. 

38.  Klamann.     ■ '  Bienenwirtscliaftliches  Centralblatt.     Hanover,  No.  18,1888. 

39.  Reports  of  the  bee  keepers'  association  of  the  Province  of  Ontario,  1890. 

40.  Planta.     <Schweizerische  Bienen-zeitung,  1893. 

41.  Howard,    W.    R.     Foul    Brood;    Its    natural  history    and     rational    treatment. 

<Chicago,  1894. 

42.  McEvoy.     Foul  Brood,  its  cause  and  cure.     <Trenton,  N.  J.,  1895. 

43.  Root,  A.  I.     <Gleanings  in  Bee  Culture,  Vol.  XXIV,  p.  853,  1896. 

44.  Cowan.     <British  Bee  Journal,  Vol.  XII,  p.  128. 

45.  Govan.     <British  Bee  Journal,  Vol.  XXIII,  p.  434. 

46.  Formalin  as  a  cure  for  foul  brood.     <Gleanings  in  Bee  Culture,  Vol.  XXX,  No. 

13,  p.  544,  1902. 

47.  Weber,  C.  H.  W.     Formalin  gas  as  a  cure  for  foul  brood.     <Cincinnati,  Ohio, 

1903. 

48.  Burri,  R.     Bakteriologische  Forschungen  uber  die  Faulbrut.     <Schweizerische 

Bienen-zeitung,  Nos.  10  and  11,  1904. 

49.  Reidenbach.     1st  das  Vernichten  der  Faulbrautstocke  das  ficherste  Mittel  zur 

Bekampfung  der  Faulbrut?     <Leipziger  Bienen-zeitung,  January,  1903. 

50.  Neumann.     Zur  Klarung  der  Faulbrutfrage.     <Ibid.,  1904. 


INDEX. 


Page. 

Acknowledgments  of  author 2 

Apiary,  diseases 30-46 

normal,  bacteria  found 12-30 

tabulation  of  micro-organisms 28 

technique  for  study  of  bacteria 7-13 

Aspergillus  pollinis,  probably  not  cause  of  ' '  pickle  brood  " 43,  45 

Bacillus  A  (B.  mesentericus  f),  description 13-14 

.     mistaken  for  Bacillus  alvei 3,  29,  33 

on  combs  in  normal  apiary 13,  29 

on  healthy  adult  honeybees 16 

alvei,  confusion  with  BaciUus  A 3,  29,  33 

description 36-37 

discovery 31 

distribution  in  infected  hives 38-39,  44-45 

European  foul  brood  produced  experimentally  by  inoculation..  37-38 

name  wrongly  given  to  other  bacteria 33-34 

not  present  in  American  foul  brood 4,  32 

present  in  European  foul  brood 3,  32,  35, 44 

so-called  "New  York  bee  disease"  or  "black  brood"  3 

relation  with  Bacillus  mesentericus  rulgatus 31 

B,  description 15-16 

in  pollen  and  intestine  of  healthy  honeybees 15,  29 

cholerx  suis,  description 21-22 

in  intestine  of  healthy  honeybee 21,  29 

cloacse,  description 19-20 

in  intestine  of  healthy  honeybee 19,  29 

coli  communis,  description 20-21 

in  intestine  of  healthy  honeybee 20,  29 

E,  description 22-23 

in  intestine  of  healthy  honeybee 22,  29 

gaytoni,  considered  by  some  as  cause  of  paralysis  of  bees 44 

larvx,  description 42-43 

formerly  termed  Bacillus  X 40 

present  in  American  foul  brood 32, 40,  42, 45 

mesentericus  f     ( See  Bacillus  A . ) 

mesentericus  vulgatus,  relation  with  Bacillus  alvei 31 

millii ,  not  found  in  so-called  ' '  black  brood  " 44 

subgastricus,  description 23-24 

in  intestine  of  healthy  honeybee 23,  29 

X=  Bacillus  larvse 40 

47 


48  INDEX. 

Page. 

Bacteria,  from  combs  of  normal  apiary 13-15 

pollen  of  normal  apiary 15-16 

in  healthy  larvae  not  usual 16,  29 

honey  from  normal  apiary  not  usual 16,  29 

intestine  of  healthy  honeybee 18-25 

of  apiary,  cultures,  how  obtained 8 

suggestions  for  description 10-12 

which  are  described  in  paper 9 

differentiation  and  identification 9 

material  for  study  how  obtained 7-8 

media  employed  for  cultures 10-12 

oxygen  requirements 10 

staining  properties 10 

technique  in  study 7-13 

variations  in  size 9-10 

of  normal  apiary 12-30 

on  healthy  adult  honeybees 16-18 

Bacterium  aridiformans,  description 14-15 

on  combs  in  normal  apiary 14 

cyaneua  {Micrococcus  cyarn  ua  I,  description 16-17 

on  combs  of  normal  apiary 29 

on  healthy  adult  bees  and  pollen 16 

D,  description 19 

in  intestine  of  healthy  honeybee 19-29 

mijcoides,  description 24 

in  intestine  of  healthy  honeybee 24,  29 

Bee  bread,  fermentation  considered  cause  of  foul  brood  formerly 31 

diseases 30-46 

history ; 30-32 

modi  lied  names  necessary 3-4 

theories  as  to  cause 30-31 

Bees,  diseased  adult,  parts  of  body  infected  by  Bacillus  alrei 39,  45 

healthy  adult,  bacteria  found  externally 16-18 

in  intestine 15, 18,  25,  29 

Saccharomyces  F  in  intestine 25,  29 

<  »vary  of  queen,  Bad/hi*  alvei  present  accidentally 39, 45 

Bibliography  to  Part  I 29-30 

II 46 

"Black  brood"  =  European  foul  brood 44,  45 

=  foul  brood  of  Cheshire  and  Cheyne 3 

occurrence  of  Bacillus  alvei 35 

origin  of  term 31, 43-44 

term  may  be  discarded 45 

Blight  of  flowers,  supposed  by  ancients  related  to  bee  diseases 30 

Brood,  diseased,  occurrence  of  Saccharomyces  G 26-27,  29 

Climatic  conditions,  as  affecting  European  foul  brood 38 

Combs,  of  healthy  apiary,  occurrence  of  bacteria 13-15 

fungi 25 

Saccharomyces  roseus 25,  26 

diseased  apiary,  occurrence  of  Bacillus  alvei 39, 45 

Cryptococcus  alvearis,  formerly  considered  cause  of  foul  brood 31 

formerly  considered  cause  of  foul  brood 31 

Cultures,  of  bacteria  of  apiary,  how  obtained 8 


INDEX.  -49 

Page. 

Cultures,  of  bacteria  of  apiary,  media  employed 10-12 

suggestions  for  description 10-12 

those  described 9 

Differentiation  of  bacteria  of  apiary 9 

Formaldehyde  gas,  insufficient  disinfectant  against  European  foul  brood  as 

ordinarily  used 39-40 

Foul  brood,  American,  application  of  term 45 

author's  investigations 41-42 

Bacillus  alvei  not  present 4,  32 

larvse  present 32,  42,  45 

confusion  with  foul  brood  of  Cheshire  and  Cheyne . .  4,  40,  45 

symptoms 40-41 

confusion  regarding  diseases 33 

disease  of  Cheshire  and  Cheyne  renamed  European  foul  brood.  3,  32,  44 

European,  application  of  term 45 

author's  investigations 34-36 

Bacillus  alvei  present 4,  32, 35,  44 

=  foul  brood  of  Cheshire  and  Cheyne 3,  32, 44 

formaldehyde  gas  insufficient  disinfectant  as  ordinarily 

used 39-40 

more  destructive  in  cool,  damp  weather 38 

produced   by  experimental  inoculation  with   Bacillus 

alvei 38 

symptoms 32-33 

of  Cheyne,  named  European  foul  brood 32 

term  applied  to  two  distinct  diseases 31-32,  45 

use  of  term  in  New  York  State 31 

Frames,  in  diseased  apiaries,  occurrence  of  Bacillus  alvei 39, 45 

Fungi,  formerly  considered  cause  of  foul  brood 31 

in  intestines  of  healthy  honeybees 25 

pollen  and  combs  of  normal  apiaries 25 

Fungus.     {See  Aspergillus  poUinis,  Cryptococcus,  Cryptococcus  alvearis,  and  Pan  - 
histophyton  ovatum. ) 

Hives,  of  diseased  apiaries,  occurrence  of  Bacillus  alvei 39,  45 

temperature  approximates  that  of  warm-blooded  animals 29 

Honey,  from  foul-brood  combs,  occurrence  of  Bacillus  alvei 39,  45 

healthy  hives,  quite  uniformly  sterile 16,  29 

Ichneumon  apium  mellificarium,  formerly  supposed  cause  of  infectious  bee  dis- 
ease           30 

Identification  of  bacteria  of  apiary 9 

Intestine  of  healthy  honeybee,  occurrence  of  bacteria 18-25 

fungi 25 

Saccharomyces  F 25,  29 

Larvse  of  honeybee  dead  from  disease,  occurrence  of  Bacillus  alvei 39,  44 

healthy  honeybee,  usually  sterile 16,  29 

Micrococcus  C,  description 17-18 

on  combs  of  healthy  honeybees 29 

healthy  adult  honeybees 17 

cyaneus.      (See  Bacterium  cyaneus.) 

Micro-organisms  normally  present  in  the  apiary,  tabulation 28 

Morphology  of  bacteria  of  apiary 9-10 

*  New  York  bee  disease. "     (See  u  Black  brood. ' ' ) 


50  INDEX. 

Page. 

Nonpathogenic  bacteria  of  honeybees,  necessity  for  study 3 

Oxygen  requirements,  of  bacteria  of  apiary 10 

Palsy.     (See  Paralysis.) 

Panhlstophyton  ovatum,  a  related  fungus,  formerly  supposed  cause  of  bee  disease.  30 

Paralysis,  of  honeybees,  cause  unknown 44,  45 

"Pickle  brood,"  Aspergillus pollinis  probably  not  cause 43,  45 

bacteriological  findings  from  author's  examinations 43 

disease  of  bees 31,43,44,45 

Pollen,  in  foul-brood  combs,  occurrence  of  Bacillus  alvei 39,  44 

healthy  combs,  occurrence  of  bacteria 15, 16 

f  nngi 25 

Propolis.     (See  Combs. ) 

Pseudomonas fluoresceins  liquefaciens,  description 24-25 

in  intestine  of  healthy  adult  honeybee  ...  24,  29 

Saccharomyce8  F,  description 25-26 

in  intestine  of  healthy  adult  honeybee 25,  29 

<i,  description 26-27 

in  dead  larvae  of  diseased  honeybees 26,  29 

in  normal  apiary 25-27 

roseus,  description 26 

in  comb  of  normal  apiary 25,  29 

Staining  properties  of  bacteria  of  apiary 10 

Summary  to  Part  I 29 

II 44-45 

Technique  in  study  of  bacteria  of  apiary 7-13 

Variations  in  size  of  bacteria  of  apiary 9-10 

Wax.     (See  Combs.) 

"  X  Brood "=  American  foul  brood 40 

o 


liifi 

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